Differentiation of pancreatic endocrine cells

ABSTRACT

Disclosed herein are compositions and methods related to differentiation of stem cells into pancreatic endocrine cells. In some aspects, the methods provided herein relate to generation of pancreatic β cell, α cell, δ cells, and EC cells in vitro. In some aspects, the disclosure provides pharmaceutical compositions including the cells generated according to the methods disclosed herein, as well as methods of treatment making use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 63/059,433, filed Jul. 31, 2020,which is hereby incorporated by reference in its entirety.

BACKGROUND

Generation of stem cell derived β-cells can provide a potentially usefulstep toward the generation of islets and pancreatic organs. One of therapidly growing diseases that may be treatable by stem cell derivedtissues is diabetes. Type 1 diabetes results from autoimmune destructionof β-cells in the pancreatic islet. Type 2 diabetes results fromperipheral tissue insulin resistance and β-cell dysfunction. Diabeticpatients, particularly those suffering from type 1 diabetes, canpotentially be cured through transplantation of new β-cells. Patientstransplanted with cadaveric human islets can be made insulin independentfor 5 years or longer via this strategy, but this approach is limitedbecause of the scarcity and quality of donor islets. Generation of anunlimited supply of human β-cells from stem cells can extend thistherapy to millions of new patients and can be an important test casefor translating stem cell biology into the clinic.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.Absent any indication otherwise, publications, patents, and patentapplications mentioned in this specification are incorporated herein byreference in their entireties.

SUMMARY

Disclosed herein, in some aspects, is a method that comprises: (a)differentiating PDX1-positive, NKX6.1-negative pancreatic progenitorcells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells bycontacting said PDX1-positive, NKX6.1-negative pancreatic progenitorcells with a ROCK inhibitor, a growth factor from TGF-β superfamily, agrowth factor from FGF family, a RA signaling pathway activator, and aSHH pathway inhibitor, thereby generating a population of cellscomprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells;(b) contacting said population of cells comprising PDX1-positive,NKX6.1-positive pancreatic progenitor cells with a first compositioncomprising a PKC activator, a γ-secretase inhibitor, a ROCK inhibitor, agrowth factor from TGFβ superfamily, a growth factor from FGF family, aRA signaling pathway activator, and a SHH pathway inhibitor, for a firsttime period; and (c) after said first time period, contacting saidpopulation of cells comprising PDX1-positive, NKX6.1-positive pancreaticprogenitor cells with a second composition comprising a PKC activator, aγ-secretase inhibitor, a TGF-β signaling pathway inhibitor, a growthfactor from EGF family, a RA signaling pathway activator, a SHH pathwayinhibitor, a TH signaling pathway activator, a protein kinase inhibitor,a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigeneticmodifying compound, for a second time period.

Disclosed herein, in some aspects, is a method that comprises: (a)contacting a population of cells comprising PDX1-positive,NKX6.1-positive pancreatic progenitor cells with a first compositioncomprising a PKC activator, a γ-secretase inhibitor, and a factorselected from the group consisting of: a ROCK inhibitor, a growth factorfrom TGFβ superfamily, a growth factor from FGF family, a RA signalingpathway activator, and a SHH pathway inhibitor, for a first time period;and (b) after said first time period, contacting said population ofcells comprising PDX1-positive, NKX6.1-positive pancreatic progenitorcells with a second composition comprising a PKC activator, aγ-secretase inhibitor, and a factor selected from the group consistingof: a TGF-β signaling pathway inhibitor, a growth factor from EGFfamily, a RA signaling pathway activator, a SHH pathway inhibitor, a THsignaling pathway activator, a protein kinase inhibitor, a ROCKinhibitor, a BMP signaling pathway inhibitor, and an epigeneticmodifying compound, for a second time period.

In some cases, the method further comprises after said second timeperiod, contacting said population of cells comprising PDX1-positive,NKX6.1-positive pancreatic progenitor cells with a third compositionthat differentiates at least some of said PDX1-positive, NKX6.1-positivepancreatic progenitor cells into NKX6.1-positive, ISL1-positiveendocrine cells, thereby generating a population of cells comprisingNKX6.1-positive, ISL1-positive endocrine cells.

Disclosed herein, in some aspects, is a method that comprises: (a)contacting a population of cells comprising PDX1-positive,NKX6.1-positive pancreatic progenitor cells with a first compositioncomprising a PKC activator and a factor selected from the groupconsisting of: a ROCK inhibitor, a growth factor from TGFβ superfamily,a growth factor from FGF family, a RA signaling pathway activator, and aSHH pathway inhibitor, for a first time period; (b) after said firsttime period, contacting said population of cells comprisingPDX1-positive, NKX6.1-positive pancreatic progenitor cells with a secondcomposition comprising a PKC activator and a factor selected from thegroup consisting of: a TGF-β signaling pathway inhibitor, a growthfactor from EGF family, a RA signaling pathway activator, a SHH pathwayinhibitor, a TH signaling pathway activator, a protein kinase inhibitor,a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigeneticmodifying compound, for a second time period; and (c) after said secondtime period, contacting said population of cells comprisingPDX1-positive, NKX6.1-positive pancreatic progenitor cells with a thirdcomposition that differentiates at least some of said PDX1-positive,NKX6.1-positive pancreatic progenitor cells into NKX6.1-positive,ISL1-positive endocrine cells, thereby generating a population of cellscomprising NKX6.1-positive, ISL1-positive endocrine cells, wherein saidpopulation of cells comprising NKX6.1-positive, ISL1-positive endocrinecells comprises: (i) an increased proportion of cells expressingglucagon; (ii) a reduced proportion of cells expressing VMAT1; (iii) anincreased proportion of cells expressing somatostatin; or (iv) anincreased proportion of cells expressing C-peptide, as compared to acorresponding population of cells which is generated without saidcontacting of said PDX1-positive, NKX6.1-positive pancreatic progenitorcells with said PKC activator in said first composition or in saidsecond composition.

In some cases, said third composition comprises a TGF-β signalingpathway inhibitor, a thyroid hormone (TH) signaling pathway activator,and an epigenetic modifying compound. In some cases, said thirdcomposition comprises a differentiation factor selected from the groupconsisting of: a TGF-β signaling pathway inhibitor, a thyroid hormonesignaling pathway activator, an epigenetic modifying compound, a growthfactor from EGF family, a RA signaling pathway activator, a SHH pathwayinhibitor, a γ-secretase inhibitor, a protein kinase inhibitor, a ROCKinhibitor, and a BMP signaling pathway inhibitor. In some cases, saidthird composition comprises said TGF-β signaling pathway inhibitor, saidthyroid hormone signaling pathway activator, said epigenetic modifyingcompound, said growth factor from EGF family, said RA signaling pathwayactivator, said SHH pathway inhibitor, said γ-secretase inhibitor, saidprotein kinase inhibitor, said ROCK inhibitor, and said BMP signalingpathway inhibitor. In some cases, said third composition does notcomprise said PKC activator. In some cases, the first compositioncomprises said ROCK inhibitor, said growth factor from TGFβ superfamily,said growth factor from FGF family, said RA signaling pathway activator,and said SHH pathway inhibitor. In some cases, the second compositioncomprises said TGF-β signaling pathway inhibitor, said growth factorfrom EGF family, said RA signaling pathway activator, said SHH pathwayinhibitor, said TH signaling pathway activator, said protein kinaseinhibitor, said ROCK inhibitor, said BMP signaling pathway inhibitor,and said epigenetic modifying compound. In some cases, said populationof cells comprising NKX6.1-positive, ISL1-positive endocrine cellscomprises: (i) an increased proportion of cells expressing somatostatin;(ii) an increased proportion of cells expressing glucagon; (iii) areduced proportion of cells expressing VMAT1; or (iv) an increasedproportion of cells expressing C-peptide, as compared to a correspondingpopulation of cells which is generated without said contacting of saidPDX1-positive, NKX6.1-positive pancreatic progenitor cells with said PKCactivator in said first composition or in said second composition. Insome cases, said population of cells comprising NKX6.1-positive,ISL1-positive endocrine cells comprises: (i) an increased proportion ofcells expressing somatostatin; (ii) an increased proportion of cellsexpressing glucagon; (iii) a reduced proportion of cells expressingVMAT1; and (iv) an increased proportion of cells expressing C-peptide,as compared to a corresponding population of cells which is generatedwithout said contacting of said PDX1-positive, NKX6.1-positivepancreatic progenitor cells with said PKC activator in said firstcomposition or in said second composition. In some cases, saidpopulation of cells comprising NKX6.1-positive, ISL1-positive endocrinecells comprises: at least about 4% cells expressing somatostatin, atleast about 15% cells expressing glucagon, at most about 35% cellsexpressing VMAT1, or at least about 40% cells expressing C-peptide, asmeasured by flow cytometry. In some cases, said population of cellscomprising NKX6.1-positive, ISL1-positive endocrine cells comprises: atleast about 100% more cells expressing somatostatin, at least about 200%more cells expressing glucagon, at least about 50% fewer cellsexpressing VMAT1, or at least about 20% more cells expressing C-peptide,as measured by flow cytometry, as compared to a corresponding populationof cells which is generated without said contacting of saidPDX1-positive, NKX6.1-positive pancreatic progenitor cells with said PKCactivator in said first composition or in said second composition. Insome cases, first time period is from one to three days. In some cases,said first time period is about two days. In some cases, said secondtime period is from one to three days. In some cases, said second timeperiod is about two days. In some cases, said PKC activator is selectedfrom the group consisting of: phorbol 12,13-dibutyrate (PDBU), FR236924, Prostratin, SC-9, and TPPB. In some cases, said PKC activatorcomprises PDBU. In some cases, said PKC activator is contacted to saidpopulation of cells comprising PDX1-positive, NKX6.1-positive pancreaticprogenitor cells at a concentration from 100 nM to 1000 nM. In somecases, said PKC activator is contacted to said population of cellscomprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells ata concentration about 500 nM. In some cases, said γ-secretase inhibitorcomprises XXI. In some cases, said γ-secretase inhibitor is contacted tosaid population of cells comprising PDX1-positive, NKX6.1-positivepancreatic progenitor cells at a concentration from 0.5 μM to 10 μM. Insome cases, said γ-secretase inhibitor is contacted to said populationof cells comprising PDX1-positive, NKX6.1-positive pancreatic progenitorcells at a concentration about 2 μM.

In some cases, the method further comprises: obtaining said populationof cells comprising PDX1-positive, NKX6.1-positive pancreatic progenitorcells by contacting a population of cells comprising PDX1-positive,NKX6.1-negative pancreatic progenitor cells with a compositioncomprising said PDX1-positive, NKX6.1-negative pancreatic progenitorcells with a ROCK inhibitor, a growth factor from TGFβ superfamily, agrowth factor from FGF family, a RA signaling pathway activator, and aSHH pathway inhibitor, and differentiating said PDX1-positive,NKX6.1-negative pancreatic progenitor cells into said PDX1-positive,NKX6.1-positive pancreatic progenitor cells. In some cases, the methodfurther comprises: differentiating FOXA2-positive, PDX1-negativeprimitive gut tube cells into said PDX1-positive, NKX6.1-negativepancreatic progenitor cells by contacting said FOXA2-positive,PDX1-negative primitive gut tube cells with a ROCK inhibitor, a growthfactor from FGF family, a BMP signaling pathway inhibitor, a PKCactivator, a retinoic acid signaling pathway activator, a SHH pathwayinhibitor, and a growth factor from TGF-β superfamily. In some cases,the method further comprises: differentiating definitive endoderm cellsinto said FOXA2-positive, PDX1-negative gut tube cells by contactingsaid definitive endoderm cells with a growth factor from FGF family.

Disclosed herein, in some aspects, is a method, comprising: (a)differentiating pluripotent stem cells in a population into definitiveendoderm cells by contacting said pluripotent stem cells with a growthfactor from TGF-β superfamily and a WNT signaling pathway activator; (b)differentiating said definitive endoderm cells into FOXA2-positive,PDX1-negative primitive gut tube cells by contacting said definitiveendoderm cells with a growth factor from FGF family; (c) differentiatingsaid FOXA2-positive, PDX1-negative primitive gut tube cells intoPDX1-positive, NKX6.1-negative pancreatic progenitor cells by contactingsaid FOXA2-positive, PDX1-negative primitive gut tube cells with a ROCKinhibitor, a growth factor from FGF family, a BMP signaling pathwayinhibitor, a PKC activator, a retinoic acid signaling pathway activator,a SHH pathway inhibitor, and a growth factor from TGF-β superfamily; (d)differentiating said PDX1-positive, NKX6.1-negative pancreaticprogenitor cells into PDX1-positive, NKX6.1-positive pancreaticprogenitor cells by contacting said PDX1-positive, NKX6.1-negativepancreatic progenitor cells with a ROCK inhibitor, a growth factor fromTGFβ superfamily, a growth factor from FGF family, a RA signalingpathway activator, and a SHH pathway inhibitor; (e) incubating saidPDX1-positive, NKX6.1-positive pancreatic progenitor cells with a firstcomposition comprising a PKC activator, a γ-secretase inhibitor, afactor selected from the group consisting of: a ROCK inhibitor, a growthfactor from TGFβ superfamily, a growth factor from FGF family, a RAsignaling pathway activator, and a SHH pathway inhibitor, for a firsttime period of one to three days; and (f) after (e), incubating saidPDX1-positive, NKX6.1-positive pancreatic progenitor cells with a secondcomposition comprising said PKC activator, said γ-secretase inhibitor, afactor selected from the group consisting of: a TGF-β signaling pathwayinhibitor, a growth factor from EGF family, a RA signaling pathwayactivator, a SHH pathway inhibitor, a TH signaling pathway activator, aprotein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathwayinhibitor, and an epigenetic modifying compound, for a second timeperiod of one to three days; (g) after (f), differentiating saidPDX1-positive, NKX6.1-positive pancreatic progenitor cells into a cellpopulation comprising NKX6.1-positive, ISL1-positive endocrine cells bycontacting said PDX1-positive, NKX6.1-positive pancreatic progenitorcells with a TGF-β signaling pathway inhibitor, a growth factor from EGFfamily, a RA signaling pathway activator, a SHH pathway inhibitor, a THsignaling pathway activator, a γ-secretase inhibitor, a protein kinaseinhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and anepigenetic modifying compound. In some cases, said SHH pathway inhibitorcomprises SANT1; said RA signaling pathway activator comprises retinoicacid; said γ-secretase inhibitor comprises XXI; said growth factor fromthe EGF family comprises betacellulin; said BMP signaling pathwayinhibitor comprises LDN or DMH; said TGF-β signaling pathway inhibitorcomprises Alk5 inhibitor II; said thyroid hormone signaling pathwayactivator comprises GC-1; said protein kinase inhibitor comprisesstaurosporine; said ROCK inhibitor comprises thiazovivin; or saidepigenetic modifying compound comprises DZNep, GSK126, or EPZ6438.

Disclosed herein, in some aspects, is a method that comprises: (a)contacting a plurality of PDX1-positive, NKX6.1-negative pancreaticprogenitor cells with one or more of a ROCK inhibitor, a growth factorfrom TGFβ superfamily, a growth factor from FGF family, a RA signalingpathway activator, and a SHH pathway inhibitor, thereby generating afirst population of cells; (b) contacting the first population of cellswith a PKC activator and a γ-secretase inhibitor and one or more of aROCK inhibitor, a growth factor from the TGFβ superfamily, a growthfactor from the FGF family, a RA signaling pathway activator, and a SHHpathway inhibitor, thereby generating a second population of cells; and(c) contacting the second population of cells with a PKC activator, aγ-secretase inhibitor and one or more of a TGF-β signaling pathwayinhibitor, a growth factor from EGF family, a RA signaling pathwayactivator, a SHH pathway inhibitor, a TH signaling pathway activator, aprotein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathwayinhibitor, and an epigenetic modifying compound, thereby generating athird population of cells.

Disclosed herein, in some aspects, is a method that comprises contactinga population of cells with a γ-secretase inhibitor and one or both of agrowth factor from the TGFβ superfamily and a growth factor from the FGFfamily. In some embodiments, the population of cells comprisesPDX1-positive cells. In some embodiments, the population of cellscomprises PDX1-positive, NKX6.1-negative cells. In some embodiments, thepopulation of cells comprises PDX1-positive, NKX6.1-positive cells.

Disclosed herein, in some aspects, is a method that comprises (a)contacting a plurality of PDX1-positive, NKX6.1-negative pancreaticprogenitor cells with one or more of a ROCK inhibitor, a growth factorfrom the TGFβ superfamily, a growth factor from the FGF family, a RAsignaling pathway activator, and a SHH pathway inhibitor, for a periodof no more than 1-5 days, thereby generating a first population ofcells; (b) contacting the first population of cells with a γ-secretaseinhibitor. In some embodiments, the contacting of step (a) is for aperiod of 4 or 5 days. In some embodiments, step (b) further comprisescontacting the first population of cells with one or more of a PKCactivator, a ROCK inhibitor, a growth factor from the TGFβ superfamily,a growth factor from the FGF family, a RA signaling pathway activator,and a SHH pathway inhibitor.

Disclosed herein, in some aspects, is a method that comprises: (a)contacting a plurality of PDX1-positive, NKX6.1-negative pancreaticprogenitor cells with one or more of a ROCK inhibitor, a growth factorfrom TGFβ superfamily, a growth factor from FGF family, a RA signalingpathway activator, and a SHH pathway inhibitor, thereby generating afirst population of cells; (b) contacting the first population of cellswith a PKC activator and one or more of a ROCK inhibitor, a growthfactor from the TGFβ superfamily, a growth factor from the FGF family, aRA signaling pathway activator, and a SHH pathway inhibitor, therebygenerating a second population of cells; wherein the PKC activator is abenzolactam-derivative; and (c) contacting the second population ofcells with the PKC activator, a γ-secretase inhibitor, and one or moreof a TGF-β signaling pathway inhibitor, a growth factor from EGF family,a RA signaling pathway activator, a SHH pathway inhibitor, a THsignaling pathway activator, a protein kinase inhibitor, a ROCKinhibitor, a BMP signaling pathway inhibitor, and an epigeneticmodifying compound, thereby generating a third population of cells.

In some cases, the benzolactam-derivative is TPPB. In some cases, step(b) further comprises contacting the first population of cells with aγ-secretase inhibitor. In some cases, the method further comprises: (d)contacting the third population of cells with one or more of a TGF-βsignaling pathway inhibitor, a RA signaling pathway activator, a THsignaling pathway activator, a protein kinase inhibitor, a ROCKinhibitor, a BMP signaling pathway inhibitor, and an epigeneticmodifying compound, thereby generating a fourth population of cells. Insome cases, step (d) does not comprise contacting the third populationof cells with a PKC activator. In some cases, step (d) does not comprisecontacting the third population of cells with a γ-secretase inhibitor.In some cases, step (d) does not comprise contacting the thirdpopulation of cells with a SHH pathway inhibitor. In some cases, step(d) does not comprise contacting the third population of cells with agrowth factor from EGF family. In some cases, the method furthercomprises: (e) contacting the fourth population of cells with one ormore of a serum albumin protein, vitamin C, a TGF-β signaling pathwayinhibitor, a SHH pathway inhibitor, a TH signaling pathway activator, aprotein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathwayinhibitor, and an epigenetic modifying compound, thereby generating afifth population of cells. In some cases, step (e) comprises contactingthe fourth population of cells with a PKC activator.

Disclosed herein, in some aspects, is a method that comprises: (a)contacting a plurality of PDX1-positive, NKX6.1-negative pancreaticprogenitor cells with one or more of a ROCK inhibitor, a growth factorfrom TGFβ superfamily, a growth factor from FGF family, a RA signalingpathway activator, and a SHH pathway inhibitor, thereby generating afirst population of cells; (b) contacting the first population of cellswith a PKC activator and one or more of a ROCK inhibitor, a growthfactor from the TGFβ superfamily, a growth factor from the FGF family, aRA signaling pathway activator, and a SHH pathway inhibitor, therebygenerating a second population of cells; (c) contacting the secondpopulation of cells with a PKC activator and one or more of aγ-secretase inhibitor, a TGF-β signaling pathway inhibitor, a growthfactor from EGF family, a RA signaling pathway activator, a SHH pathwayinhibitor, a TH signaling pathway activator, a protein kinase inhibitor,a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigeneticmodifying compound, thereby generating a third population of cells; (d)contacting the third population of cells with one or more of a TGF-βsignaling pathway inhibitor, a RA signaling pathway activator, a THsignaling pathway activator, a protein kinase inhibitor, a ROCKinhibitor, a BMP signaling pathway inhibitor, and an epigeneticmodifying compound, thereby generating a fourth population of cells; and(e) contacting the fourth population of cells with a PKC activator andone or more of a serum albumin protein, vitamin C, a TGF-β signalingpathway inhibitor, a SHH pathway inhibitor, a TH signaling pathwayactivator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signalingpathway inhibitor, and an epigenetic modifying compound, therebygenerating a fifth population of cells.

In some cases, step (e) comprises contacting the fourth population ofcells with a serum albumin protein. In some cases, step (a) is performedover the course of 1, 2, 3, 4, 5 or 6 days. In some cases, step (a) isperformed over the course of 3-5 days (e.g., 4 days). In some cases,step (b) is performed over the course of 1, 2, 3 or 4 days. In somecases, step (b) is performed over the course of 1-3 days (e.g., 2 days).In some cases, step (c) is performed over the course of 1, 2, 3, or 4days. In some cases, step (c) is performed over the course of 1-3 days(e.g., 2 days). In some cases, step (d) is performed over the course of1, 2, 3, 4, 5, 6, or 7 days. In some cases, step (d) is performed overthe course of 4-6 days (e.g., 5 days). In some cases, step (e) isperformed over the course of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 days. In some cases, step (e) is performed over the course of10-12 days. In some cases, the first population of cells comprisesPDX1-positive, NKX6.1-negative cells and/or PDX1-positive,NKX6.1-positive cells. In some cases, the second population of cellscomprises PDX1-positive and NKX6.1-positive cells. In some cases, thethird population of cells comprises PDX1-positive, NKX6.1-positive,ISL1-negative cells and/or PDX1-positive, NKX6.1-positive, ISL1-positivecells. In some cases, the fourth population of cells comprisesPDX1-positive, NKX6.1-positive, ISL1-positive cells. In some cases, thefifth population of cells comprises cells that express C-peptide andISL1 but not VMAT1. In some cases, 30-90%, 30-80%, 30-70%, 30-60%,30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%,50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-80%,or 80-90% of the cells in the fourth population of cells expressC-peptide and ISL1 but not VMAT1. In some cases, 40-60% of the cells inthe fourth population of cells express C-peptide and ISL1 but not VMAT1.In some cases, the fourth population of cells comprises cells thatexpress glucagon but not somatostatin. In some cases, 5-40%, 5-35%,5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%,10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%,20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35% or 35-40% of thecells in the fourth population of cells express glucagon but notsomatostatin. In some cases, 10-25% of the cells in the fourthpopulation of cells express somatostatin but not glucagon. In somecases, the fourth population of cells comprises cells that expresssomatostatin but not glucagon. In some cases, 3-20%, 3-15%, 3-12%,3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%,5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%,8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or15-20% of the cells in the fourth population of cells expresssomatostatin but not glucagon. In some cases, step (a) comprisescontacting a plurality of PDX1-positive, NKX6.1-negative pancreaticprogenitor cells with a ROCK inhibitor, a growth factor from TGFβsuperfamily, a growth factor from FGF family, a RA signaling pathwayactivator, and a SHH pathway inhibitor. In some cases, step (b)comprises contacting the first population of cells with a ROCKinhibitor, a growth factor from the TGFβ superfamily, a growth factorfrom the FGF family, a RA signaling pathway activator, and a SHH pathwayinhibitor. In some cases, step (c) comprises contacting the secondpopulation of cells with a gamma-secretase inhibitor, a TGF-β signalingpathway inhibitor, a growth factor from EGF family, a RA signalingpathway activator, a SHH pathway inhibitor, a TH signaling pathwayactivator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signalingpathway inhibitor, and an epigenetic modifying compound. In some cases,step (d) comprises contacting the third population of cells with serumalbumin protein, a TGF-β signaling pathway inhibitor, a SHH pathwayinhibitor, a TH signaling pathway activator, a protein kinase inhibitor,a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigeneticmodifying compound. In some cases, the ROCK inhibitor for use in step(a), (b), (c), (d), and/or (e) is thiazovavin or Y-27632. In some cases,the growth factor from the TGFβ superfamily for use in steps (a) and/or(b) is activin A. In some cases, the growth factor from the FGF familyfor use in steps (a) and/or (b) is KGF. In some cases, the RA signalingpathway activator for use in steps (a), (b) and/or (c) is retinoic acid.In some cases, the SHH pathway inhibitor for use in steps (a), (b)and/or (c) is Sant-1. In some cases, the PKC activator for use in steps(b), (c) and/or (d) is selected from the group consisting of: phorbol12,13-dibutyrate (PDBU), FR 236924, Prostratin, SC-9, and TPPB. In somecases, the PKC activator is PDBU. In some cases, the γ-secretaseinhibitor for use in step (b) and/or (c) is XXI. In some cases, theTGF-β signaling pathway inhibitor for use in step (c), (d), and/or (e)is ALK5i. In some cases, the growth factor from the EGF family for usein step (c) is betacellulin. In some cases, the TH signaling pathwayactivator for use in step (c), (d), and/or (e) is T3, GC-1 or a thyroidhormone derivative. In some cases, the protein kinase inhibitor for usein step (c), (d), and/or (e) is staurosporine. In some cases, the BMPsignaling pathway inhibitor for use in step (c), (d), and/or (e) isLDN193189 or DMH-1. In some cases, the epigenetic modifying compound foruse in step (c), (d), and/or (e) is DZNep.

Disclosed herein, in some aspects, is an in vitro composition thatcomprises PDX1-positive, NKX6.1-positive pancreatic progenitor cells;NKX6.1-positive, ISL1-positive endocrine cells; a PKC activator; and aγ-secretase inhibitor.

Disclosed herein, in some aspects, is an in vitro composition thatcomprises PDX1-positive, NKX6.1-negative pancreatic progenitor cells;PDX1-positive, NKX6.1-positive pancreatic progenitor cells; a PKCactivator; and a γ-secretase inhibitor. In some embodiments, at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the cells in thecomposition are PDX1-positive, NKX6.1-positive pancreatic progenitorcells. In some embodiments, less than 90%, 80%, 70%, 60%, 50%, 40%, 30%,20%, or 10% of the cells in the composition are PDX1-positive,NKX6.1-negative pancreatic progenitor cells.

In some embodiments of the composition, said PKC activator is selectedfrom the group consisting of: phorbol 12,13-dibutyrate (PDBU), FR236924, Prostratin, SC-9, and TPPB. In some cases, the γ-secretaseinhibitor is DAPT(N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester).In some cases, the γ-secretase inhibitor is XXI.

In some embodiments of the composition, the composition furthercomprises a growth factor from the FGF family. In some embodiments, thegrowth factor from the FGF family is KGF. In some embodiments, thecomposition further comprises a growth factor of the TGFβ superfamily.In some embodiments, the growth factor of the TGFβ superfamily isactivin A.

Disclosed herein, in some aspects, is an in vitro composition thatcomprises PDX1-positive, NKX6.1-positive pancreatic progenitor cells;NKX6.1-positive, ISL1-positive endocrine cells; and a PKC activator;wherein the PKC activator is a benzolactam derivative.

In some embodiments of the composition, the PKC activator is TPPB. Insome cases, the composition further comprises a γ-secretase inhibitor.In some cases, the γ-secretase inhibitor is DAPT. In some cases, theγ-secretase inhibitor is XXI. In some cases, the composition furthercomprises a differentiation factor selected from the group consistingof: a TGF-β signaling pathway inhibitor, a thyroid hormone signalingpathway activator, an epigenetic modifying compound, a growth factorfrom EGF family, a RA signaling pathway activator, a SHH pathwayinhibitor, a protein kinase inhibitor, a ROCK inhibitor, and a BMPsignaling pathway inhibitor. In some cases, the composition furthercomprises serum albumin protein. In some cases, the composition furthercomprises serum albumin protein, a TGF-β signaling pathway inhibitor, athyroid hormone signaling pathway activator, an epigenetic modifyingcompound, a SHH pathway inhibitor, a protein kinase inhibitor, a ROCKinhibitor, and a BMP signaling pathway inhibitor. In some cases, theROCK inhibitor is thiazovavin. In some cases, the RA signaling pathwayactivator is retinoic acid. In some cases, the SHH pathway inhibitor isSant-1. In some cases, the TGF-β signaling pathway inhibitor is ALK5i.In some cases, the growth factor from the EGF family is betacellulin. Insome cases, the thyroid hormone signaling pathway activator is T3, GC-1or a thyroid hormone derivative. In some cases, the protein kinaseinhibitor is staurosporine. In some cases, the BMP signaling pathwayinhibitor is LDN193189 or DMH-1. In some cases, the epigenetic modifyingcompound is DZNep.

Disclosed herein, in some aspects, is a composition that comprises an invitro cell population, wherein said cell population comprises: (a) atleast about 35% cells expressing C-peptide and not expressing VMAT1; and(b) at most about 35% cells expressing VMAT1, or at least about 15%cells expressing glucagon (e.g., as measured by flow cytometry). In someaspects, the disclosure provides a composition that comprises an invitro cell population, wherein said cell population comprises: at leastabout 35% cells expressing C-peptide and not expressing VMAT1; and (i)at most about 35% cells expressing VMAT1, and/or (ii) at least about 15%cells expressing glucagon. In some embodiments, the percentages of cellsare measured by flow cytometry.

In some cases, said cell population comprises at most about 30% cellsexpressing VMAT1 and at least about 20% cells expressing glucagon. Insome cases, said cell population comprises at most about 30% cellsexpressing VMAT1 and at least about 20% cells expressing glucagon, asmeasured by flow cytometry. In some cases, said cell populationcomprises at least about 15% cells expressing glucagon and notexpressing somatostatin. In some cases, said cell population comprisesat least about 4% cells expressing somatostatin and not expressingglucagon.

Disclosed herein, in some aspects, is a composition that comprises apopulation of cells, wherein: a) 30-90%, 30-80%, 30-70%, 30-60%, 30-50%,30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%,50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-80%, or80-90% of the cells in the population of cells express C-peptide andISL1 but not VMAT1; b) 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%,10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%,15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%,30-40%, 30-35% or 35-40% of the cells in the population of cells expressglucagon but not somatostatin; and/or c) 3-20%, 3-15%, 3-12%, 3-10%,3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%,5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%,8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or 15-20% ofthe cells in the population of cells express somatostatin but notglucagon.

Disclosed herein, in some aspects, is a composition that comprises apopulation of cells, wherein: a) 30-90%, 30-80%, 30-70%, 30-60%, 30-50%,30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%,50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-80%, or80-90% of the cells in the population of cells express C-peptide andISL1 but not VMAT1; b) 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%,10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%,15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%,30-40%, 30-35% or 35-40% of the cells in the population of cells expressglucagon but not somatostatin; and c) 3-20%, 3-15%, 3-12%, 3-10%, 3-8%,3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%,5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%,8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or 15-20% ofthe cells in the population of cells express somatostatin but notglucagon.

Disclosed herein, in some aspects, is a composition that comprises apopulation of cells, wherein: a) 5-35%, 5-30%, 5-25%, 5-20%, 5-15%,10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-35%, 15-30%, 15-25%, 15-20%,20-35%, 20-30%, 20-25%, 25-35%, 25-30%, or 30-35%, of the cells in thepopulation of cells express VMAT1 but not C-peptide; b) 5-40%, 5-35%,5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%,10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%,20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35% or 35-40% of thecells in the population of cells express glucagon but not somatostatin;and/or c) 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%,4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%,7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%,10-12%, 10-15%, 12-20%, 12-15% or 15-20% of the cells in the populationof cells express somatostatin but not glucagon.

Disclosed herein, in some aspects, is a composition that comprises apopulation of cells, wherein: a) 5-35%, 5-30%, 5-25%, 5-20%, 5-15%,10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-35%, 15-30%, 15-25%, 15-20%,20-35%, 20-30%, 20-25%, 25-35%, 25-30%, or 30-35%, of the cells in thepopulation of cells express VMAT1 but not C-peptide; b) 5-40%, 5-35%,5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%,10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%,20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35% or 35-40% of thecells in the population of cells express glucagon but not somatostatin;and c) 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%,4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%,7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%,10-12%, 10-15%, 12-20%, 12-15% or 15-20% of the cells in the populationof cells express somatostatin but not glucagon.

In some embodiments of the composition, 30-90%, 30-80%, 30-70%, 30-60%,30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%,50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-80%,or 80-90% of the cells in the population of cells express C-peptide andISL1 but not VMAT1.

In some embodiments of the composition, 40-60% of the cells in thepopulation of cells express C-peptide and ISL1 but not VMAT1; 10-25%, ofthe cells in the population of cells express glucagon but notsomatostatin; and 4-10% of the cells in the population of cells expresssomatostatin but not glucagon. In some cases, less than 25%, less than20%, less than 18%, less than 15%, less than 12%, or less than 10% ofthe cells in the population of cells express VMAT1 but not C-peptide. Insome cases, the population of cells are generated from stem cells invitro. In some cases, the cells expressing C-peptide and not expressingVMAT1 exhibit glucose-stimulated insulin secretion response in vitro. Insome cases, secretion of insulin by the cells expressing C-peptide andnot expressing VMAT1 in response to a glucose challenge is proportionalto glucose concentration of the glucose challenge. In some cases, thecells expressing C-peptide and not expressing VMAT1 secrete insulin inresponse to one or more glucose challenges. In some cases, the cellsexpressing C-peptide and not expressing VMAT1 secrete insulin inresponse to a first glucose challenge, a second glucose challenge, and athird glucose challenge, wherein the first glucose challenge, the secondglucose challenge, and the third glucose challenge are appliedsequentially.

Disclosed herein, in some aspects, is an in vitro composition comprisingPDX1-positive cells, a γ-secretase inhibitor, and one or both of agrowth factor from the TGFβ superfamily and a growth factor from the FGFfamily. In some embodiments, the composition of cells comprisesPDX1-positive, NKX6.1-negative cells. In some embodiments, thecomposition of cells comprises PDX1-positive, NKX6.1-positive cells.

In some embodiments, the composition further comprises any one of orcombination of a PKC activator, a growth factor from the FGF family, aROCK inhibitor, a growth factor from the TGFβ superfamily, a sonichedgehog pathway inhibitor, and a retinoic acid signaling pathwayactivator.

Disclosed herein, in some aspects, is an in vitro composition comprisingPDX1-positive, NKX6.1-negative pancreatic progenitor cells;PDX1-positive, NKX6.1-positive pancreatic progenitor cells; and aγ-secretase inhibitor. In some embodiments, the γ-secretase inhibitor isXXI.

In some embodiments, at least 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least90% of the cells in the composition are PDX1-positive, NKX6.1-positivepancreatic progenitor cells. In some embodiments, less than 90%, lessthan 80%, less than 70%, less than 60%, less than 50%, less than 40%,less than 30%, less than 20%, or less than 10% of the cells in thecomposition are PDX1-positive, NKX6.1-negative pancreatic progenitorcells.

In some embodiments, the composition further comprises a growth factorfrom the FGF family. In some embodiments, the composition furthercomprises a sonic hedgehog pathway inhibitor. In some embodiments, thecomposition further comprises a ROCK inhibitor. In some embodiments, thecomposition further comprises a growth factor from the TGFβ superfamily.In some embodiments, the composition further comprises a retinoic acidsignaling pathway activator. In some embodiments, the compositionfurther comprises a PKC activator.

In some embodiments, the composition further comprises any two of a PKCactivator, a growth factor from the FGF family, a ROCK inhibitor, agrowth factor from the TGFβ superfamily, a sonic hedgehog pathwayinhibitor, and a retinoic acid signaling pathway activator. In someembodiments, the composition further comprises any three of a PKCactivator, a growth factor from the FGF family, a ROCK inhibitor, agrowth factor from the TGFβ superfamily, a sonic hedgehog pathwayinhibitor, and a retinoic acid signaling pathway activator. In someembodiments, the composition further comprises any four of a PKCactivator, a growth factor from the FGF family, a ROCK inhibitor, agrowth factor from the TGFβ superfamily, a sonic hedgehog pathwayinhibitor, and a retinoic acid signaling pathway activator. In someembodiments, the composition further comprises any five of a PKCactivator, a growth factor from the FGF family, a ROCK inhibitor, agrowth factor from the TGFβ superfamily, a sonic hedgehog pathwayinhibitor, and a retinoic acid signaling pathway activator. In someembodiments, the composition further comprises any six of a PKCactivator, a growth factor from the FGF family, a ROCK inhibitor, agrowth factor from the TGFβ superfamily, a sonic hedgehog pathwayinhibitor, and a retinoic acid signaling pathway activator.

In some embodiments of the composition, the growth factor from the FGFfamily is KGF. In some embodiments, the sonic hedgehog pathway inhibitoris SANT-1. In some embodiments, the ROCK inhibitor is thiazovivin. Insome embodiments, the growth factor from the TGFβ superfamily is activinA. In some embodiments, the retinoic acid signaling pathway activator isretinoic acid. In some embodiments, the PKC activator is PDBU.

Disclosed herein, in some aspects, is a population of in vitrodifferentiated cells comprising NKX6.1-positive, ISL1-positive cells andNKX6.1-negative, ISL1-positive cells; wherein the population comprisesmore NKX6.1-negative, ISL1-positive cells than NKX6.1-positive,ISL1-positive cells; and wherein at least 73% of the cells in thepopulation are ISL1-positive cells. In some embodiments, less than 12%of the cells in the population are NKX6.1-negative, ISL1-negative cells.

Disclosed herein, in some aspects, is a population of in vitrodifferentiated cells comprising NKX6.1-positive, ISL1-positive cells andNKX6.1-negative, ISL1-positive cells; wherein at least 40% of the cellsin the population are NKX6.1-negative, ISL1-positive cells. In someembodiments, less than 12% of the cells in the population areNKX6.1-negative, ISL1-negative cells.

Disclosed herein, in some aspects, is a population of in vitrodifferentiated cells comprising NKX6.1-positive, ISL1-positive cells andNKX6.1-negative, ISL1-positive cells; and wherein less than 12% of thecells in the population are NKX6.1-negative, ISL1-negative cells.

In some embodiments, less than 10%, less than 8%, less than 6%, or lessthan 4% of the cells in the population are NKX6.1-negative,ISL1-negative cells. In some embodiments, at least 60%, at least 65%, atleast 70%, at least 73%, at least 75%, or at least 80% of the cells inthe population are ISL1-positive cells. In some embodiments, 2-12%,4-12%, 6-12%, 8-12%, 2-8%, 4-8%, 3-6% or 3-5% of the cells in thepopulation are NKX6.1-negative, ISL1-negative cells. In someembodiments, 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-60%, 60-90%,60-85%, 60-80%, 60-75%, 60-70%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%,70-90%, 70-85%, 70-80%, 70-75%, 75-90%, 75-85%, 75-80%, 80-90%, 80-85%,or 85-90% of the cells in the population are ISL1-positive cells.

In some embodiments, the population comprises more NKX6.1-negative,ISL1-positive cells than NKX6.1-positive, ISL1-positive cells. In someembodiments, at least 40% of the cells in the population areNKX6.1-negative, ISL1-positive cells. In some embodiments, at least 45%,at least 50%, about 40-50%, about 45-55%, or about 50-55% of the cellsin the population are NKX6.1-negative, ISL1-positive cells. In someembodiments, at least 74%, at least 75%, at least 80%, at least 85%, atleast 90%, about 85-95%, or about 90-95% of the cells in the populationare ISL1-positive cells.

In some embodiments, the population comprises more stem cell-derivedalpha cells than stem cell-derived beta cells. In some embodiments, thepopulation of cells is derived from stem cells in vitro.

In some embodiments, the population further comprises a medium. In someembodiments, the medium comprises a sugar. In some embodiments, thesugar is sucrose or glucose. In some embodiments, the medium comprisesthe sugar at a concentration of between about 0.05% and about 1.5%. Insome embodiments, the medium is a CMRL medium; or wherein the medium isHypoThermosol® FRS Preservation Media.

In some embodiments, the population of cells is in a cell cluster. Insome embodiments, the population of cells are in one or more cellcluster. In some embodiments, the cell cluster is between about 125 andabout 225 microns in diameter, between about 130 and about 160 micronsin diameter, between about 170 and about 225 microns in diameter,between about 140 and about 200 microns in diameter, between about 140and about 170 microns in diameter, between about 160 and about 220microns in diameter, between about 170 and about 215 microns indiameter, or between about 170 and about 200 microns in diameter.

In some embodiments, the population has a genetic disruption in thebeta-2-microglobulin gene.

In some embodiments, the population comprises NKX6.1-positive,ISL1-positive cells that express lower levels of MAFA thanNKX6.1-positive, ISL1-positive cells from the pancreas of a healthycontrol adult subject. In some embodiments, the population comprisesNKX6.1-positive, ISL1-positive cells that express higher levels of MAFBthan NKX6.1-positive, ISL1-positive cells from the pancreas of a healthycontrol adult subject. In some embodiments, the population comprisesNKX6.1-positive, ISL1-positive cells that express higher levels of SIX2,HOPX, IAPP and/or UCN3 than NKX6.1-positive, ISL1-positive cells fromthe pancreas of a healthy control adult subject.

In some embodiments, the population comprises NKX6.1-positive,ISL1-positive cells that do not express MAFA. In some embodiments, thepopulation comprises NKX6.1-positive, ISL1-positive cells that expressMAFB.

In some embodiments, the population is contained in a device forimplantation into a subject. In some aspects, the present disclosureprovides an implantable encapsulation device comprising the population.In some embodiments, the device has been implanted in a subject havingdiabetes. In some embodiments, the subject has Type I Diabetes. In someaspects, the present disclosure provides a method of treating a subject,the method comprising administering to the subject a compositioncomprising the population, or implanting in the subject the device.

Disclosed herein, in some aspects, is a pharmaceutical composition thatcomprises the composition disclosed herein, or the cell populationproduced according to the method disclosed herein, and apharmaceutically acceptable excipient or carrier.

Disclosed herein, in some aspects, is a device that comprises thecomposition disclosed herein, or the cell population produced accordingto the method disclosed herein, wherein the device is configured toproduce and release insulin when implanted into a subject.

Disclosed herein, in some aspects, is a method of treating a subjectthat comprises administering the subject with the composition disclosedhereinv, or the cell population produced according to the methoddisclosed herein, or the device disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the present disclosure are set forth with particularityin the appended claims. A better understanding of the features andadvantages of the present will be obtained by reference to the followingdetailed description that sets forth illustrative embodiments, in whichthe principles of the disclosure are utilized, and the accompanyingdrawings of which:

FIG. 1 shows single-cell sequencing results of in vitro endocrine cellpopulations generated according to two exemplary differentiationprotocols (Version A and Version B), with or without PDBU applied onS4d5 to S5d2.

FIGS. 2A-2B summarize the percentage of C-peptide-positive,VMAT1-negative cells (FIG. 2A) in the in vitro endocrine cellpopulations generated according to two exemplary differentiationprotocols, with or without PDBU applied on S4d5 to S5d2, as measured byflow cytometry (FIG. 2B).

FIG. 3 summarizes the percentage of glucagon-positive,somatostatin-negative cells (GCG+/SST−) in the in vitro endocrine cellpopulations generated according to two exemplary differentiationprotocols, with or without PDBU applied on S4d5 to S5d2.

FIG. 4 summarizes the percentage of somatostatin-positive,glucagon-negative cells (SST+/GCG−) in the in vitro endocrine cellpopulations generated according to two exemplary differentiationprotocols, with or without PDBU applied on S4d5 to S5d2.

FIG. 5 summarizes the percentage of VMAT1-positive, C-peptide-negativecells (VMAT1+/c-peptide−) in the in vitro endocrine cell populationsgenerated according to two exemplary differentiation protocols, with orwithout PDBU applied on S4d5 to S5d2.

FIGS. 6A-6B summarize the percentage of SOX9-positive cells before (FIG.6A) and after reaggregation (FIG. 6B) in the in vitro endocrine cellpopulations generated according to two exemplary differentiationprotocols, with or without PDBU applied on S4d5 to S5d2, as measured byflow cytometry.

FIG. 7 summarizes the recovery ratio after reaggregation in the in vitroendocrine cell populations generated according to two exemplarydifferentiation protocols, with or without PDBU applied on S4d5 to S5d2.

FIG. 8 summarizes glucose-stimulated insulin secretion (GSIS) responseof the in vitro endocrine cell populations generated according to twoexemplary differentiation protocols, with or without PDBU applied onS4d5 to S5d2.

FIG. 9 summarizes the insulin content of the in vitro endocrine cellpopulations generated according to two exemplary differentiationprotocols, with or without PDBU applied on S4d5 to S5d2.

FIGS. 10A-10B summarize the percentage of NKX6.1-positive, ISL1-positivecells (FIG. 10B) in the in vitro cell populations generated according tothree exemplary differentiation protocols, with or without PDBU or PDBUand XXI applied during S4d5 to S5d2, as measured by flow cytometry (FIG.10A).

FIGS. 11A-11C summarize the percentage of NKX6.1-positive/negative andISL1-positive/negative cells (FIG. 11B) in the in vitro cell populationsgenerated according to three exemplary differentiation protocols: a)Version A without PDBU or TPPB (Version A); b) with PDBU (VA/PDBU); orc) with TPPB (VA/TPPB), as measured by flow cytometry (FIG. 11A). FIG.11C shows the cell yield for Version A, VA/PDBU, VA/TPPB, as well asVA/TPPB+XXI.

DETAILED DESCRIPTION

The following description and examples illustrate embodiments of thepresent disclosure in detail. It is to be understood that thisdisclosure is not limited to the particular embodiments described hereinand as such can vary. Those of skill in the art will recognize thatthere are numerous variations and modifications of this disclosure,which are encompassed within its scope.

All terms are intended to be understood as they would be understood by aperson skilled in the art. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which the disclosurepertains.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Although various features of the present disclosure can be described inthe context of a single embodiment, the features can also be providedseparately or in any suitable combination. Conversely, although thepresent disclosure can be described herein in the context of separateembodiments for clarity, the present disclosure can also be implementedin a single embodiment.

The following definitions supplement those in the art and are directedto the current application and are not to be imputed to any related orunrelated case, e.g., to any commonly owned patent or application.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice for testing of the presentdisclosure, the preferred materials and methods are described herein.Accordingly, the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. It must be noted that, as used in thespecification, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

In this application, the use of “or” means “and/or” unless statedotherwise. The terms “and/or” and “any combination thereof” and theirgrammatical equivalents as used herein, can be used interchangeably.These terms can convey that any combination is specificallycontemplated. Solely for illustrative purposes, the following phrases“A, B, and/or C” or “A, B, C, or any combination thereof” can mean “Aindividually; B individually; C individually; A and B; B and C; A and C;and A, B, and C.” The term “or” can be used conjunctively ordisjunctively, unless the context specifically refers to a disjunctiveuse.

Furthermore, use of the term “including” as well as other forms, such as“include”, “includes,” and “included,” is not limiting.

Reference in the specification to “some embodiments,” “an embodiment,”“one embodiment” or “other embodiments” means that a particular feature,structure, or characteristic described in connection with theembodiments is included in at least some embodiments, but notnecessarily all embodiments, of the present disclosures.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps. It is contemplated that any embodimentdiscussed in this specification can be implemented with respect to anymethod or composition of the present disclosure, and vice versa.Furthermore, compositions of the present disclosure can be used toachieve methods of the present disclosure.

The term “about” in relation to a reference numerical value and itsgrammatical equivalents as used herein can include the numerical valueitself and a range of values plus or minus 10% from that numericalvalue.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, up to 10%, up to 5%, or up to 1% of a given value. In anotherexample, the amount “about 10” includes 10 and any amounts from 9 to 11.In yet another example, the term “about” in relation to a referencenumerical value can also include a range of values plus or minus 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. Alternatively,particularly with respect to biological systems or processes, the term“about” can mean within an order of magnitude, preferably within 5-fold,and more preferably within 2-fold, of a value. Where particular valuesare described in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

The term “diabetes” and its grammatical equivalents as used herein canrefer to is a disease characterized by high blood sugar levels over aprolonged period. For example, the term “diabetes” and its grammaticalequivalents as used herein can refer to all or any type of diabetes,including, but not limited to, type 1, type 2, cystic fibrosis-related,surgical, gestational diabetes, and mitochondrial diabetes. In somecases, diabetes can be a form of hereditary diabetes.

The term “endocrine cell(s),” if not particularly specified, can referto hormone-producing cells present in the pancreas of an organism, suchas “islet”, “islet cells”, “islet equivalent”, “islet-like cells”,“pancreatic islets” and their grammatical equivalents. In an embodiment,the endocrine cells can be differentiated from pancreatic progenitorcells or precursors. Islet cells can comprise different types of cells,including, but not limited to, pancreatic α cells, pancreatic β cells,pancreatic δ cells, pancreatic F cells, and/or pancreatic ε cells. Isletcells can also refer to a group of cells, cell clusters, or the like.

The terms “progenitor” and “precursor” cell are used interchangeablyherein and refer to cells that have a cellular phenotype that is moreprimitive (e.g., is at an earlier step along a developmental pathway orprogression than is a fully differentiated cell) relative to a cellwhich it can give rise to by differentiation. Often, progenitor cellscan also have significant or very high proliferative potential.Progenitor cells can give rise to multiple distinct differentiated celltypes or to a single differentiated cell type, depending on thedevelopmental pathway and on the environment in which the cells developand differentiate.

A “precursor thereof” as the term related to an insulin-positiveendocrine cell can refer to any cell that is capable of differentiatinginto an insulin-positive endocrine cell, including for example, apluripotent stem cell, a definitive endoderm cell, a primitive gut tubecell, a pancreatic progenitor cell, or endocrine progenitor cell, whencultured under conditions suitable for differentiating the precursorcell into the insulin-positive endocrine cell.

The terms “stem cell-derived β cell,” “SC-β cell,” “functional β cell,”“functional pancreatic β cell,” “mature SC-β cell,” and theirgrammatical equivalents can refer to cells (e.g., non-native pancreaticβ cells) that display at least one marker indicative of a pancreatic βcell (e.g., PDX-1 or NKX6.1), expresses insulin, and display a glucosestimulated insulin secretion (GSIS) response characteristic of anendogenous mature β cell. In some embodiments, the terms “SC-β cell” and“non-native β cell” as used herein are interchangeable. In someembodiments, the “SC-β cell” comprises a mature pancreatic cell. It isto be understood that the SC-β cells need not be derived (e.g.,directly) from stem cells, as the methods of the disclosure are capableof deriving SC-β cells from any insulin-positive endocrine cell orprecursor thereof using any cell as a starting point (e.g., one can useembryonic stem cells, induced-pluripotent stem cells, progenitor cells,partially reprogrammed somatic cells (e.g., a somatic cell which hasbeen partially reprogrammed to an intermediate state between an inducedpluripotent stem cell and the somatic cell from which it was derived),multipotent cells, totipotent cells, a transdifferentiated version ofany of the foregoing cells, etc., as the invention is not intended to belimited in this manner). In some embodiments, the SC-β cells exhibit aresponse to multiple glucose challenges (e.g., at least one, at leasttwo, or at least three or more sequential glucose challenges). In someembodiments, the response resembles the response of endogenous islets(e.g., human islets) to multiple glucose challenges. In someembodiments, the morphology of the SC-β cell resembles the morphology ofan endogenous β cell. In some embodiments, the SC-β cell exhibits an invitro GSIS response that resembles the GSIS response of an endogenous βcell. In some embodiments, the SC-β cell exhibits an in vivo GSISresponse that resembles the GSIS response of an endogenous β cell. Insome embodiments, the SC-β cell exhibits both an in vitro and in vivoGSIS response that resembles the GSIS response of an endogenous β cell.The GSIS response of the SC-β cell can be observed within two weeks oftransplantation of the SC-β cell into a host (e.g., a human or animal).In some embodiments, the SC-β cells package insulin into secretorygranules. In some embodiments, the SC-β cells exhibit encapsulatedcrystalline insulin granules. In some embodiments, the SC-β cellsexhibit a stimulation index of greater than 1. In some embodiments, theSC-β cells exhibit a stimulation index of greater than 1.1. In someembodiments, the SC-β cells exhibit a stimulation index of greater than2. In some embodiments, the SC-β cells exhibit cytokine-inducedapoptosis in response to cytokines. In some embodiments, insulinsecretion from the SC-β cells is enhanced in response to knownantidiabetic drugs (e.g., secretagogues). In some embodiments, the SC-βcells are monohormonal. In some embodiments, the SC-β cells do notabnormally co-express other hormones, such as glucagon, somatostatin orpancreatic polypeptide. In some embodiments, the SC-β cells exhibit alow rate of replication. In some embodiments, the SC-β cells increaseintracellular Ca2+ in response to glucose.

The terms “stem cell-derived α cell,” “SC-α cell,” “functional α cell,”“functional pancreatic α cell,” “mature SC-α cell,” and theirgrammatical equivalents can refer to cells (e.g., non-native pancreaticα cells) that display at least one marker indicative of a pancreatic αcell (e.g., glucagon, expressing ISL1 but not NKX6.1), expressesglucagon, and secretes functional glucagon. In some embodiments, the“SC-α cell” does not express somatostatin. In some embodiments, the“SC-α cell” does not express insulin. In some embodiments, the terms“SC-α cell” and “non-native α cell” as used herein are interchangeable.In some embodiments, the “SC-α cell” comprises a mature pancreatic cell.

The terms “stem cell-derived δ cell,” “SC-δ cell,” “functional δ cell,”“functional pancreatic δ cell,” “mature SC-δ cell,” and theirgrammatical equivalents can refer to cells (e.g., non-native pancreaticδ cells) that display at least one marker indicative of a pancreatic δcell (e.g., somatostatin), expresses and secretes somatostatin. In someembodiments, “SC-δ cell” does not express glucagon. In some embodiments,“SC-δ cell” does not express insulin. In some embodiments, the terms“SC-δ cell” and “non-native δ cell” as used herein are interchangeable.In some embodiments, the “SC-δ cell” comprises a mature pancreatic cell.

The terms “stem cell-derived enterochromaffin (EC) cell,” “SC-EC cell,”and their grammatical equivalents can refer to cells (e.g., non-nativepancreatic EC cells) that display at least one marker indicative of apancreatic EC cell (e.g., VMAT1 (vesicular monoamine transporter 1),expressing NKX6.1 but not ISL1). In some embodiments, the terms “SC-ECcell” and “non-native EC cell” as used herein are interchangeable.

Similar to SC-β cells, it is to be understood that the SC-α, SC-δ cells,and SC-EC cells need not be derived (e.g., directly) from stem cells, asthe methods of the disclosure are capable of deriving SC-α cells fromother precursor cells generated during in vitro differentiation of SC-βcells as a starting point (e.g., one can use embryonic stem cells,induced-pluripotent stem cells, progenitor cells, partially reprogrammedsomatic cells (e.g., a somatic cell which has been partiallyreprogrammed to an intermediate state between an induced pluripotentstem cell and the somatic cell from which it was derived), multipotentcells, totipotent cells, a transdifferentiated version of any of theforegoing cells, etc., as the invention is not intended to be limited inthis manner).

As used herein, the term “insulin producing cell” and its grammaticalequivalent refer to a cell differentiated from a pancreatic progenitor,or precursor thereof, which secretes insulin. An insulin-producing cellcan include pancreatic β cell as that term is described herein, as wellas pancreatic β-like cells (e.g., insulin-positive, endocrine cells)that synthesize (e.g., transcribe the insulin gene, translate theproinsulin mRNA, and modify the proinsulin mRNA into the insulinprotein), express (e.g., manifest the phenotypic trait carried by theinsulin gene), or secrete (release insulin into the extracellular space)insulin in a constitutive or inducible manner. A population of insulinproducing cells e.g., produced by differentiating insulin-positiveendocrine cells or a precursor thereof into SC-β cells according to themethods of the present disclosure can be pancreatic β cell or (β-likecells (e.g., cells that have at least one, or at least two least two)characteristic of an endogenous β cell and exhibit a glucose stimulatedinsulin secretion (GSIS) response that resembles an endogenous adult βcell. The population of insulin-producing cells, e.g. produced by themethods as disclosed herein can comprise mature pancreatic β cell orSC-β cells, and can also contain non-insulin-producing cells (e.g.,cells of cell like phenotype with the exception they do not produce orsecrete insulin).

The terms “insulin-positive β-like cell,” “insulin-positive endocrinecell,” and their grammatical equivalents can refer to cells (e.g.,pancreatic endocrine cells) that displays at least one marker indicativeof a pancreatic β cell and also expresses insulin but lack a glucosestimulated insulin secretion (GSIS) response characteristic of anendogenous β cell. Exemplary markers of “insulin-positive endocrinecell” include, but not limited to, NKX6.1 (NK6 homeobox 1), ISL1(Isletl), and insulin. In some cases, the terms “insulin-positiveendocrine cell” and “NKX6.1-positive, ISL1-positive cell” are usedinterchangeably.

The term “β cell marker” refers to, without limitation, proteins,peptides, nucleic acids, polymorphism of proteins and nucleic acids,splice variants, fragments of proteins or nucleic acids, elements, andother analyte which are specifically expressed or present in pancreaticβ cells. Exemplary β cell markers include, but are not limited to,pancreatic and duodenal homeobox 1 (PDX1) polypeptide, insulin,c-peptide, amylin, E-cadherin, Hnf3β, PCI/3, B2, Nkx2.2, GLUT2, PC2,ZnT-8, ISL1, Pax6, Pax4, NeuroD, 1 Inf1b, Hnf-6, Hnf-3beta, and MafA,and those described in Zhang et al., Diabetes. 50(10):2231-6 (2001). Insome embodiment, the β cell marker is a nuclear β-cell marker. In someembodiments, the β cell marker is PDX1 or PH3.

The term “pancreatic endocrine marker” can refer to without limitation,proteins, peptides, nucleic acids, polymorphism of proteins and nucleicacids, splice variants, fragments of proteins or nucleic acids,elements, and other analyte which are specifically expressed or presentin pancreatic endocrine cells. Exemplary pancreatic endocrine cellmarkers include, but are not limited to, Ngn-3, NeuroD and Islet-1.

The term “pancreatic progenitor,” “pancreatic endocrine progenitor,”“pancreatic precursor,” “pancreatic endocrine precursor” and theirgrammatical equivalents are used interchangeably herein and can refer toa stem cell which is capable of becoming a pancreatic hormone expressingcell capable of forming pancreatic endocrine cells, pancreatic exocrinecells or pancreatic duct cells. These cells are committed todifferentiating towards at least one type of pancreatic cell, e.g. βcells that produce insulin; α cells that produce glucagon; δ cells (or Dcells) that produce somatostatin; and/or F cells that produce pancreaticpolypeptide. Such cells can express at least one of the followingmarkers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

The term “PDX1-positive pancreatic progenitor” as used herein can referto a cell which is a pancreatic endoderm (PE) cell which has thecapacity to differentiate into SC-β cells, such as pancreatic β cells. APDX1-positive pancreatic progenitor expresses the marker PDX1. Othermarkers include, but are not limited to Cdcp1, or Ptf1a, or HNF6 orNRx2.2. The expression of PDX1 may be assessed by any method known bythe skilled person such as immunochemistry using an anti-PDX1 antibodyor quantitative RT-PCR. In some cases, a PDX1-positive pancreaticprogenitor cell lacks expression of NKX6.1. In some cases, aPDX1-positive pancreatic progenitor cell can also be referred to asPDX1-positive, NKX6.1-negative pancreatic progenitor cell due to itslack of expression of NKX6.1. In some cases, the PDX1-positivepancreatic progenitor cells can also be termed as “pancreatic foregutendoderm cells.”

The terms “PDX1-positive, NKX6.1-positive pancreatic progenitor,” and“NKX6.1-positive pancreatic progenitor” are used interchangeably hereinand can refer to a cell which is a pancreatic endoderm (PE) cell whichhas the capacity to differentiate into insulin-producing cells, such aspancreatic β cells. A PDX1-positive, NKX6.1-positive pancreaticprogenitor expresses the markers PDX1 and NKX6-1. Other markers include,but are not limited to Cdcp1, or Ptf1a, or HNF6 or NRx2.2. Theexpression of NKX6-1 may be assessed by any method known by the skilledperson such as immunochemistry using an anti-NKX6-1 antibody orquantitative RT-PCR. As used herein, the terms “NKX6.1” and “NKX6-1” areequivalent and interchangeable. In some cases, the PDX1-positive,NKX6.1-positive pancreatic progenitor cells can also be termed as“pancreatic foregut precursor cells.”

The terms “NeuroD” and “NeuroD1” are used interchangeably and identify aprotein expressed in pancreatic endocrine progenitor cells and the geneencoding it.

The term “epigenetics” refers to heritable changes in gene function thatdo not involve changes in the DNA sequence. Epigenetics most oftendenotes changes in a chromosome that affect gene activity andexpression, but can also be used to describe any heritable phenotypicchange that does not derive from a modification of the genome. Sucheffects on cellular and physiological phenotypic traits can result fromexternal or environmental factors, or be part of normal developmentalprogram. Epigenetics can also refer to functionally relevant changes tothe genome that do not involve a change in the nucleotide sequence.Examples of mechanisms that produce such changes are DNA methylation andhistone modification, each of which alters how genes are expressedwithout altering the underlying DNA sequence. Gene expression can becontrolled through the action of repressor proteins that attach tosilencer regions of the DNA. These epigenetic changes can last throughcell divisions for the duration of the cell's life, and can also lastfor multiple generations even though they do not involve changes in theunderlying DNA sequence of the organism. One example of an epigeneticchange in eukaryotic biology is the process of cellular differentiation.During morphogenesis, totipotent stem cells become the variouspluripotent cells, which in turn can become fully differentiated cells.

The term “epigenetic modifying compound” refers to a chemical compoundthat can make epigenetic changes genes, i.e., change gene expression(s)without changing DNA sequences. Epigenetic changes can help determinewhether genes are turned on or off and can influence the production ofproteins in certain cells, e.g., beta-cells. Epigenetic modifications,such as DNA methylation and histone modification, alter DNAaccessibility and chromatin structure, thereby regulating patterns ofgene expression. These processes are crucial to normal development anddifferentiation of distinct cell lineages in the adult organism. Theycan be modified by exogenous influences, and, as such, can contribute toor be the result of environmental alterations of phenotype orpathophenotype. Importantly, epigenetic modification has a crucial rolein the regulation of pluripotency genes, which become inactivated duringdifferentiation. Non-limiting exemplary epigenetic modifying compoundinclude a DNA methylation inhibitor, a histone acetyltransferaseinhibitor, a histone deacetylase inhibitor, a histone methyltransferaseinhibitor, a bromodomain inhibitor, or any combination thereof.

The term “differentiated cell” or its grammatical equivalents is meantany primary cell that is not, in its native form, pluripotent as thatterm is defined herein. Stated another way, the term “differentiatedcell” can refer to a cell of a more specialized cell type derived from acell of a less specialized cell type (e.g., a stem cell such as aninduced pluripotent stem cell) in a cellular differentiation process.Without wishing to be limited to theory, a pluripotent stem cell in thecourse of normal ontogeny can differentiate first to an endoderm cellthat is capable of forming pancreas cells and other endoderm cell types.Further differentiation of an endoderm cell leads to the pancreaticpathway, where ^(˜)98% of the cells become exocrine, ductular, or matrixcells, and ˜2% become endocrine cells. Early endocrine cells are isletprogenitors, which can then differentiate further into insulin-producingcells (e.g. functional endocrine cells) which secrete insulin, glucagon,somatostatin, or pancreatic polypeptide. Endoderm cells can also bedifferentiated into other cells of endodermal origin, e.g. lung, liver,intestine, thymus etc.

As used herein, the term “somatic cell” can refer to any cells formingthe body of an organism, as opposed to germline cells. In mammals,germline cells (also known as “gametes”) are the spermatozoa and ovawhich fuse during fertilization to produce a cell called a zygote, fromwhich the entire mammalian embryo develops. Every other cell type in themammalian body—apart from the sperm and ova, the cells from which theyare made (gametocytes) and undifferentiated stem cells—is a somaticcell: internal organs, skin, bones, blood, and connective tissue are allmade up of somatic cells. In some embodiments the somatic cell is a“non-embryonic somatic cell”, by which is meant a somatic cell that isnot present in or obtained from an embryo and does not result fromproliferation of such a cell in vitro. In some embodiments the somaticcell is an “adult somatic cell”, by which is meant a cell that ispresent in or obtained from an organism other than an embryo or a fetusor results from proliferation of such a cell in vitro. Unless otherwiseindicated the methods for converting at least one insulin-positiveendocrine cell or precursor thereof to an insulin-producing, glucoseresponsive cell can be performed both in vivo and in vitro (where invivo is practiced when at least one insulin-positive endocrine cell orprecursor thereof are present within a subject, and where in vitro ispracticed using an isolated at least one insulin-positive endocrine cellor precursor thereof maintained in culture).

As used herein, the term “adult cell” can refer to a cell foundthroughout the body after embryonic development.

The term “endoderm cell” as used herein can refer to a cell which isfrom one of the three primary germ cell layers in the very early embryo(the other two germ cell layers are the mesoderm and ectoderm). Theendoderm is the innermost of the three layers. An endoderm celldifferentiates to give rise first to the embryonic gut and then to thelinings of the respiratory and digestive tracts (e.g. the intestine),the liver and the pancreas.

The term “a cell of endoderm origin” as used herein can refer to anycell which has developed or differentiated from an endoderm cell. Forexample, a cell of endoderm origin includes cells of the liver, lung,pancreas, thymus, intestine, stomach and thyroid. Without wishing to bebound by theory, liver and pancreas progenitors (also referred to aspancreatic progenitors) are develop from endoderm cells in the embryonicforegut. Shortly after their specification, liver and pancreasprogenitors rapidly acquire markedly different cellular functions andregenerative capacities. These changes are elicited by inductive signalsand genetic regulatory factors that are highly conserved amongvertebrates. Interest in the development and regeneration of the organshas been fueled by the intense need for hepatocytes and pancreatic βcells in the therapeutic treatment of liver failure and type I diabetes.Studies in diverse model organisms and humans have revealedevolutionarily conserved inductive signals and transcription factornetworks that elicit the differentiation of liver and pancreatic cellsand provide guidance for how to promote hepatocyte and β celldifferentiation from diverse stem and progenitor cell types.

The term “definitive endoderm” as used herein can refer to a celldifferentiated from an endoderm cell and which can be differentiatedinto a SC-β cell (e.g., a pancreatic β cell). A definitive endoderm cellexpresses the marker Sox17. Other markers characteristic of definitiveendoderm cells include, but are not limited to MIXL2, GATA4, HNF3b, GSC,FGF17, VWF, CALCR, FOXQ1, CXCR4, Cerberus, OTX2, goosecoid, C-Kit, CD99,CMKOR1 and CRIP1. In particular, definitive endoderm cells hereinexpress Sox17 and in some embodiments Sox17 and HNF3B, and do notexpress significant levels of GATA4, SPARC, APF or DAB. Definitiveendoderm cells are not positive for the marker PDX1 (e.g. they arePDX1-negative). Definitive endoderm cells have the capacity todifferentiate into cells including those of the liver, lung, pancreas,thymus, intestine, stomach and thyroid. The expression of Sox17 andother markers of definitive endoderm may be assessed by any method knownby the skilled person such as immunochemistry, e.g., using an anti-Sox17antibody, or quantitative RT-PCR.

The term “pancreatic endoderm” can refer to a cell of endoderm originwhich is capable of differentiating into multiple pancreatic lineages,including pancreatic β cells, but no longer has the capacity todifferentiate into non-pancreatic lineages.

The term “primitive gut tube cell” or “gut tube cell” as used herein canrefer to a cell differentiated from an endoderm cell and which can bedifferentiated into a SC-β cell (e.g., a pancreatic β cell). A primitivegut tube cell expresses at least one of the following markers: HNP1-β,HNF3-β or HNF4-α. In some cases, a primitive gut tube cell isFOXA2-positive and SOX2-positive, i.e., express both FOXA2 (also knownas HNF3-β ) and SOX2. In some cases, a primitive gut tube cell isFOXA2-positive and PDX1-negative, i.e., express FOXA2 but not PDX1.Primitive gut tube cells have the capacity to differentiate into cellsincluding those of the lung, liver, pancreas, stomach, and intestine.The expression of HNF1-β and other markers of primitive gut tube may beassessed by any method known by the skilled person such asimmunochemistry, e.g., using an anti-HNF1-β antibody.

The term “stem cell” as used herein, can refer to an undifferentiatedcell which is capable of proliferation and giving rise to moreprogenitor cells having the ability to generate a large number of mothercells that can in turn give rise to differentiated, or differentiabledaughter cells. The daughter cells themselves can be induced toproliferate and produce progeny that subsequently differentiate into oneor more mature cell types, while also retaining one or more cells withparental developmental potential. The term “stem cell” can refer to asubset of progenitors that have the capacity or potential, underparticular circumstances, to differentiate to a more specialized ordifferentiated phenotype, and which retains the capacity, under certaincircumstances, to proliferate without substantially differentiating. Inone embodiment, the term stem cell refers generally to a naturallyoccurring mother cell whose descendants (progeny) specialize, often indifferent directions, by differentiation, e.g., by acquiring completelyindividual characters, as occurs in progressive diversification ofembryonic cells and tissues. Cellular differentiation is a complexprocess typically occurring through many cell divisions. Adifferentiated cell may derive from a multipotent cell which itself isderived from a multipotent cell, and so on. While each of thesemultipotent cells may be considered stem cells, the range of cell typeseach can give rise to may vary considerably. Some differentiated cellsalso have the capacity to give rise to cells of greater developmentalpotential. Such capacity may be natural or may be induced artificiallyupon treatment with various factors. In many biological instances, stemcells are also “multipotent” because they can produce progeny of morethan one distinct cell type, but this is not required for “stem-ness.”Self-renewal is the other classical part of the stem cell definition,and it is essential as used in this document. In theory, self-renewalcan occur by either of two major mechanisms. Stem cells may divideasymmetrically, with one daughter retaining the stem state and the otherdaughter expressing some distinct other specific function and phenotype.Alternatively, some of the stem cells in a population can dividesymmetrically into two stems, thus maintaining some stem cells in thepopulation as a whole, while other cells in the population give rise todifferentiated progeny only. Formally, it is possible that cells thatbegin as stem cells might proceed toward a differentiated phenotype, butthen “reverse” and re-express the stem cell phenotype, a term oftenreferred to as “dedifferentiation” or “reprogramming” or“retro-differentiation” by persons of ordinary skill in the art. As usedherein, the term “pluripotent stem cell” includes embryonic stem cells,induced pluripotent stem cells, placental stem cells, etc.

The term “pluripotent” as used herein can refer to a cell with thecapacity, under different conditions, to differentiate to more than onedifferentiated cell type, and preferably to differentiate to cell typescharacteristic of all three germ cell layers. Pluripotent cells arecharacterized primarily by their ability to differentiate to more thanone cell type, preferably to all three germ layers, using, for example,a nude mouse teratoma formation assay. Pluripotency is also evidenced bythe expression of embryonic stem (ES) cell markers, although thepreferred test for pluripotency is the demonstration of the capacity todifferentiate into cells of each of the three germ layers. It should benoted that simply culturing such cells does not, on its own, render thempluripotent. Reprogrammed pluripotent cells (e.g. iPS cells as that termis defined herein) also have the characteristic of the capacity ofextended passaging without loss of growth potential, relative to primarycell parents, which generally have capacity for only a limited number ofdivisions in culture.

As used herein, the terms “iPS cell” and “induced pluripotent stem cell”are used interchangeably and can refer to a pluripotent stem cellartificially derived (e.g., induced or by complete reversal) from anon-pluripotent cell, typically an adult somatic cell, for example, byinducing a forced expression of one or more genes.

The term “phenotype” can refer to one or a number of total biologicalcharacteristics that define the cell or organism under a particular setof environmental conditions and factors, regardless of the actualgenotype.

The terms “subject,” “patient,” or “individual” are used interchangeablyherein, and can refer to an animal, for example, a human from whom cellscan be obtained and/or to whom treatment, including prophylactictreatment, with the cells as described herein, is provided. Fortreatment of those infections, conditions or disease states which arespecific for a specific animal such as a human subject, the term subjectcan refer to that specific animal. The “non-human animals” and“non-human mammals” as used interchangeably herein, includes mammalssuch as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, andnon-human primates. The term “subject” also encompasses any vertebrateincluding but not limited to mammals, reptiles, amphibians and fish.However, advantageously, the subject is a mammal such as a human, orother mammals such as a domesticated mammal, e.g., dog, cat, horse, andthe like, or production mammal, e.g. cow, sheep, pig, and the like.“Patient in need thereof” or “subject in need thereof” is referred toherein as a patient diagnosed with or suspected of having a disease ordisorder, for instance, but not restricted to diabetes.

“Administering” used herein can refer to providing one or morecompositions described herein to a patient or a subject. By way ofexample and not limitation, composition administration, e.g., injection,can be performed by intravenous (i.v.) injection, sub-cutaneous (s.c.)injection, intradermal (i.d.) injection, intraperitoneal (i.p.)injection, or intramuscular (i.m.) injection. One or more such routescan be employed. Parenteral administration can be, for example, by bolusinjection or by gradual perfusion over time. Alternatively, orconcurrently, administration can be by the oral route. Additionally,administration can also be by surgical deposition of a bolus or pelletof cells, or positioning of a medical device. In an embodiment, acomposition of the present disclosure can comprise engineered cells orhost cells expressing nucleic acid sequences described herein, or avector comprising at least one nucleic acid sequence described herein,in an amount that is effective to treat or prevent proliferativedisorders. A pharmaceutical composition can comprise the cell populationas described herein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions can comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

Some numerical values disclosed throughout are referred to as, forexample, “X is at least or at least about 100; or 200 [or any numericalnumber].” This numerical value includes the number itself and all of thefollowing:

i) X is at least 100;

ii) X is at least 200;

iii) X is at least about 100; and

iv) X is at least about 200.

All these different combinations are contemplated by the numericalvalues disclosed throughout. All disclosed numerical values should beinterpreted in this manner, whether it refers to an administration of atherapeutic agent or referring to days, months, years, weight, dosageamounts, etc., unless otherwise specifically indicated to the contrary.

The ranges disclosed throughout are sometimes referred to as, forexample, “X is administered on or on about day 1 to 2; or 2 to 3 [or anynumerical range].” This range includes the numbers themselves (e.g., theendpoints of the range) and all of the following:

i) X being administered on between day 1 and day 2;

ii) X being administered on between day 2 and day 3;

iii) X being administered on between about day 1 and day 2;

iv) X being administered on between about day 2 and day 3;

v) X being administered on between day 1 and about day 2;

vi) X being administered on between day 2 and about day 3;

vii) X being administered on between about day 1 and about day 2; and

viii) X being administered on between about day 2 and about day 3.

All these different combinations are contemplated by the rangesdisclosed throughout. All disclosed ranges should be interpreted in thismanner, whether it refers to an administration of a therapeutic agent orreferring to days, months, years, weight, dosage amounts, etc., unlessotherwise specifically indicated to the contrary.

In aspects, the present disclosure provides compositions and methods ofdifferentiating pancreatic progenitor cells. The compositions andmethods provided herein can, in some embodiments, offer pancreatic βcells, cell populations, or cell clusters that have high purity ofpancreatic I cells, high insulin content, superior glucose-dependentinsulin secretion response, as well as appropriate percentage ofpancreatic α and δ cells and enterochromaffin cells, which can resemblenative pancreatic islets both structurally and functionally.

In some aspects, provided herein is a method of differentiatingpancreatic endocrine cells. In some cases, the method leads togeneration of increased pancreatic β cells, increased pancreatic αcells, increased pancreatic δ cells, reduced enterochromaffin cells (ECcells), or any combination thereof. In some cases, the method results ingeneration of an in vitro cell composition comprising about 30%-40%pancreatic β cells, 30%-40% pancreatic α cells, 3-10% pancreatic δcells, and/or less than 20% EC cells. In some cases, the cellcomposition generated according to the method disclosed herein hasimproved glucose-stimulated insulin secretion (GSIS) response ascompared to cell compositions generated according to conventionalmethods. In some cases, the cell composition disclosed herein hasdynamic GSIS response close to native pancreatic islets.

In some aspects, the methods provided herein take advantage of PKCactivation during or after induction of NKX6.1 expression inPDX1-positive pancreatic progenitor cells, e.g., at the end stage ofdifferentiating PDX1-positive pancreatic progenitor cells intoPDX1-positive, NKX6.1-positive pancreatic progenitor cells. Withoutbeing bound by a certain theory, activation of PKC signaling inPDX1-positive, NKX6.1-positive pancreatic progenitor cells can affectthe differentiation fate of certain cells, leading to increasedpercentage of pancreatic α cells and reduced percentage of EC cells.

In some aspects, the present disclosure provides a method thatcomprises: (a) contacting a plurality of PDX1-positive, NKX6.1-negativepancreatic progenitor cells with one or more of a ROCK inhibitor, agrowth factor from TGFβ superfamily, a growth factor from FGF family, aRA signaling pathway activator, and a SHH pathway inhibitor, therebygenerating a first population of cells; (b) contacting the firstpopulation of cells with a PKC activator and a γ-secretase inhibitor andone or more of a ROCK inhibitor, a growth factor from the TGFβsuperfamily, a growth factor from the FGF family, a RA signaling pathwayactivator, and a SHH pathway inhibitor, thereby generating a secondpopulation of cells; and (c) contacting the second population of cellswith a PKC activator, a γ-secretase inhibitor and one or more of a TGF-βsignaling pathway inhibitor, a growth factor from EGF family, a RAsignaling pathway activator, a SHH pathway inhibitor, a TH signalingpathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMPsignaling pathway inhibitor, and an epigenetic modifying compound,thereby generating a third population of cells.

In some aspects, the present disclosure provides a method comprising:contacting a population of cells with a γ-secretase inhibitor and one orboth of a growth factor from the TGFβ superfamily and a growth factorfrom the FGF family. In some embodiments, the population of cellscomprises PDX1-positive cells. In some embodiments, the population ofcells comprises PDX1-positive, NKX6.1-negative cells. In someembodiments, the population of cells comprises PDX1-positive,NKX6.1-positive cells.

In some aspects, the present disclosure provides a method comprising:(a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreaticprogenitor cells with one or more of a ROCK inhibitor, a growth factorfrom the TGFβ superfamily, a growth factor from the FGF family, a RAsignaling pathway activator, and a SHH pathway inhibitor, for a periodof no more than 1-5 days, thereby generating a first population ofcells; (b) contacting the first population of cells with a γ-secretaseinhibitor. In some embodiments, the contacting of step (a) is for aperiod of 4 or 5 days. In some embodiments, step (b) further comprisescontacting the first population of cells with one or more of a PKCactivator, a ROCK inhibitor, a growth factor from the TGFβ superfamily,a growth factor from the FGF family, a RA signaling pathway activator,and a SHH pathway inhibitor.

In some aspects, the present disclosure provides a method, comprising:(a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreaticprogenitor cells with one or more of a ROCK inhibitor, a growth factorfrom TGFβ superfamily, a growth factor from FGF family, a RA signalingpathway activator, and a SHH pathway inhibitor, thereby generating afirst population of cells; (b) contacting the first population of cellswith a PKC activator and one or more of a ROCK inhibitor, a growthfactor from the TGFβ superfamily, a growth factor from the FGF family, aRA signaling pathway activator, and a SHH pathway inhibitor, therebygenerating a second population of cells; wherein the PKC activator is abenzolactam-derivative; and (c) contacting the second population ofcells with the PKC activator, a γ-secretase inhibitor and one or more ofa TGF-β signaling pathway inhibitor, a growth factor from EGF family, aRA signaling pathway activator, a SHH pathway inhibitor, a TH signalingpathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMPsignaling pathway inhibitor, and an epigenetic modifying compound,thereby generating a third population of cells. In some cases, thebenzolactam-derivative is TPPB.

In some aspects, the present discloure provides a method that comprises:(a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreaticprogenitor cells with one or more of a ROCK inhibitor, a growth factorfrom TGFβ superfamily, a growth factor from FGF family, a RA signalingpathway activator, and a SHH pathway inhibitor, thereby generating afirst population of cells; (b) contacting the first population of cellswith a PKC activator and one or more of a ROCK inhibitor, a growthfactor from the TGFβ superfamily, a growth factor from the FGF family, aRA signaling pathway activator, and a SHH pathway inhibitor, therebygenerating a second population of cells; (c) contacting the secondpopulation of cells with a PKC activator and one or more of aγ-secretase inhibitor, a TGF-β signaling pathway inhibitor, a growthfactor from EGF family, a RA signaling pathway activator, a SHH pathwayinhibitor, a TH signaling pathway activator, a protein kinase inhibitor,a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigeneticmodifying compound, thereby generating a third population of cells; (d)contacting the third population of cells with one or more of a TGF-βsignaling pathway inhibitor, a RA signaling pathway activator, a THsignaling pathway activator, a protein kinase inhibitor, a ROCKinhibitor, a BMP signaling pathway inhibitor, and an epigeneticmodifying compound, thereby generating a fourth population of cells; and(e) contacting the fourth population of cells with a PKC activator andone or more of a serum albumin protein, vitamin C, a TGF-β signalingpathway inhibitor, a SHH pathway inhibitor, a TH signaling pathwayactivator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signalingpathway inhibitor, and an epigenetic modifying compound, therebygenerating a fifth population of cells.

In some aspects, the method disclosed herein comprises differentiatingPDX1-positive pancreatic progenitor cells into PDX1-positive,NKX6.1-positive pancreatic progenitor cells by contacting saidPDX1-positive pancreatic progenitor cells with a ROCK inhibitor, agrowth factor from TGFβ superfamily, a growth factor from FGF family, aRA signaling pathway activator, and a SHH pathway inhibitor, therebygenerating a population of cells comprising PDX1-positive,NKX6.1-positive pancreatic progenitor cells. In some cases, the methodcomprises contacting the population of cells comprising PDX1-positive,NKX6.1-positive pancreatic progenitor cells with a first compositioncomprising a PKC activator, a γ-secretase inhibitor, a ROCK inhibitor, agrowth factor from TGFβ superfamily, a growth factor from FGF family, aRA signaling pathway activator, and a SHH pathway inhibitor, for a firsttime period. In some cases, the method comprises after the first timeperiod, contacting the population of cells comprising PDX1-positive,NKX6.1-positive pancreatic progenitor cells with a second compositioncomprising the PKC activator, the γ-secretase inhibitor, a TGF-βsignaling pathway inhibitor, a growth factor from EGF family, a RAsignaling pathway activator, a SHH pathway inhibitor, a TH signalingpathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMPsignaling pathway inhibitor, and an epigenetic modifying compound, for asecond time period. In some cases, the method comprises after the secondtime period, contacting the population of cells comprisingPDX1-positive, NKX6.1-positive pancreatic progenitor cells with a thirdcomposition that differentiates at least some of the PDX1-positive,NKX6.1-positive pancreatic progenitor cells into NKX6.1-positive,ISL1-positive endocrine cells, thereby generating a population of cellscomprising NKX6.1-positive, ISL1-positive endocrine cells.

In some cases, provided herein is an in vitro composition comprising αcell population, wherein the cell population comprises: (a) at leastabout 35% cells expressing C-peptide and not expressing VMAT1; and (b)at most about 35% cells expressing VMAT1, or at least about 15% cellsexpressing glucagon (e.g., as measured by flow cytometry). In someaspects, the disclosure provides a composition that comprises an invitro cell population, wherein said cell population comprises: at leastabout 35% cells expressing C-peptide and not expressing VMAT1; and (i)at most about 35% cells expressing VMAT1, and/or (ii) at least about 15%cells expressing glucagon. In some embodiments, the percentages of cellsare measured by flow cytometry. In some cases, said cell populationcomprises at most about 30% cells expressing VMAT1 and at least about20% cells expressing glucagon.

In some cases, provided herein is an in vitro composition, comprisingPDX1-positive, NKX6.1-positive pancreatic progenitor cells;NKX6.1-positive, ISL1-positive endocrine cells; and a PKC activator;wherein the PKC activator is a benzolactam derivative.

In some cases, provided herein is a composition comprising a populationof cells, wherein: (a) 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%,40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%,60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-80%, or 80-90% of thecells in the population of cells express C-peptide and ISL1 but notVMAT1; (b) 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%,10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%,15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%,30-35% or 35-40% of the cells in the population of cells expressglucagon but not somatostatin; and/or (c) 3-20%, 3-15%, 3-12%, 3-10%,3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%,5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%,8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or 15-20% ofthe cells in the population of cells express somatostatin but notglucagon.

In some cases, provided herein is a composition comprising a populationof cells, wherein: (a) 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%,40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%,60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-80%, or 80-90% of thecells in the population of cells express C-peptide and ISL1 but notVMAT1; (b) 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%,10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%,15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%,30-35% or 35-40% of the cells in the population of cells expressglucagon but not somatostatin; and (c) 3-20%, 3-15%, 3-12%, 3-10%, 3-8%,3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%,5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%,8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or 15-20% ofthe cells in the population of cells express somatostatin but notglucagon.

In some cases, the methods provided herein include PKC activation whendifferentiating PDX1-positive, NKX6.1-positive pancreatic progenitorcells into NKX6.1-positive, ISL1-positive endocrine cells. For instance,PKC activator can be introduced at an early stage of a time period whenPDX1-positive, NKX6.1-positive pancreatic progenitor cells are contactedwith differentiation factors that direct the differentiation of thePDX1-positive, NKX6.1-positive pancreatic progenitor cells intoNKX6.1-positive, ISL1-positive endocrine cells. In some cases, themethod comprises (a) contacting a population of cells comprisingPDX1-positive, NKX6.1-positive pancreatic progenitor cells with a firstcomposition comprising the PKC activator, a ROCK inhibitor, a growthfactor from TGFβ superfamily, a growth factor from FGF family, a RAsignaling pathway activator, and a SHH pathway inhibitor, for one to twodays, thereby obtaining a first transformation cell populationcomprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells;and (b) contacting the first transformation cell population comprisingPDX1-positive, NKX6.1-positive pancreatic progenitor cells with a secondcomposition comprising the PKC activator, a TGF-β signaling pathwayinhibitor, a thyroid hormone signaling pathway activator, and anepigenetic modifying compound, for one to two days, thereby obtaining asecond transformation cell population comprising NKX6.1-positive,ISL1-positive endocrine cells.

Methods of Generating Endocrine Cells

In aspects, the present disclosure relates to compositions and methodsof generating endocrine cells from pancreatic progenitor cells orprecursors. Certain exemplary detailed protocols of generating endocrinecells to provide at least one SC-β cell are described in U.S. PatentApplication Publication No. US20150240212 and US20150218522, each ofwhich is herein incorporated by reference in its entirety.

In some cases, the method of generating a population of endocrine cellsleads to increased percentage of pancreatic a and/orb cells anddecreased percentage of pancreatic EC cells when generating pancreatic βcells. In some embodiments, the methods disclosed herein may be used toobtain an enriched population of α cells. In some embodiments, themethods disclosed herein may be used to obtain an enriched population ofδ cells. In some cases, a method for generating a population ofendocrine cells comprises (a) contacting a population of cellscomprising PDX1-positive, NKX6.1-positive pancreatic progenitor cellswith a PKC activator for a first time period; and (b) after the firsttime period, contacting the population of cells comprisingPDX1-positive, NKX6.1-positive pancreatic progenitor cells with acomposition comprising a TGF-β signaling pathway inhibitor, a thyroidhormone signaling pathway activator, and an epigenetic modifyingcompound, thereby generating a population of cells comprising pancreaticendocrine cells. In some cases, the population of cells generatedaccording to the method disclosed herein has: (i) an increasedproportion of cells expressing somatostatin; (ii) an increasedproportion of cells expressing glucagon; (iii) a reduced proportion ofcells expressing VMAT1; or (iv) an increased proportion of cellsexpressing C-peptide, as compared to a corresponding population of cellswhich is generated without contacting of the PDX1-positive,NKX6.1-positive pancreatic progenitor cells with the PKC activator forthe first time period.

In some cases, the method includes contacting the population of cellscomprising PDX1-positive, NKX6.1-positive pancreatic progenitor cellswith a first composition comprising a PKC activator, a γ-secretaseinhibitor, and a factor selected from the group consisting of: a ROCKinhibitor, a growth factor from TGFβ superfamily, a growth factor fromFGF family, a RA signaling pathway activator, and a SHH pathwayinhibitor, for a first time period; after the first time period,contacting the population of cells comprising PDX1-positive,NKX6.1-positive pancreatic progenitor cells with a second compositioncomprising the PKC activator, the γ-secretase inhibitor, and a factorselected from the group consisting of: a TGF-β signaling pathwayinhibitor, a growth factor from EGF family, a RA signaling pathwayactivator, a SHH pathway inhibitor, a TH signaling pathway activator, aprotein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathwayinhibitor, and an epigenetic modifying compound, for a second timeperiod. In some cases, the first composition comprises PKC activator, aγ-secretase inhibitor, a ROCK inhibitor, a growth factor from TGFβsuperfamily, a growth factor from FGF family, a RA signaling pathwayactivator, and a SHH pathway inhibitor. In some cases, the secondcomposition comprises the PKC activator, the γ-secretase inhibitor, aTGF-β signaling pathway inhibitor, a growth factor from EGF family, a RAsignaling pathway activator, a SHH pathway inhibitor, a TH signalingpathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMPsignaling pathway inhibitor, and an epigenetic modifying compound.

In some cases, the composition that differentiates at least some of thePDX1-positive, NKX6.1-positive pancreatic progenitor cells intoNKX6.1-positive, ISL1-positive endocrine cells comprises adifferentiation factor selected from the group consisting of: a TGF-βsignaling pathway inhibitor, a thyroid hormone signaling pathwayactivator, an epigenetic modifying compound, a growth factor from EGFfamily, a RA signaling pathway activator, a SHH pathway inhibitor, aγ-secretase inhibitor, a protein kinase inhibitor, a ROCK inhibitor, anda BMP signaling pathway inhibitor. In some cases, the compositioncomprises a TGF-β signaling pathway inhibitor, a thyroid hormonesignaling pathway activator, an epigenetic modifying compound, a growthfactor from EGF family, a RA signaling pathway activator, a SHH pathwayinhibitor, a γ-secretase inhibitor, a protein kinase inhibitor, a ROCKinhibitor, and a BMP signaling pathway inhibitor.

In some cases, the method further comprises contacting PDX1-positive,NKX6.1-positive pancreatic progenitor cells with a compositioncomprising a PKC activator. For instance, the method comprises: (a)contacting a population of cells comprising PDX1-positive,NKX6.1-positive pancreatic progenitor cells with a first compositioncomprising the PKC activator, a ROCK inhibitor, a growth factor fromTGFβ superfamily, a growth factor from FGF family, a RA signalingpathway activator, and a SHH pathway inhibitor, for one to two days,thereby obtaining a first transformation cell population comprisingPDX1-positive, NKX6.1-positive pancreatic progenitor cells; and (b)contacting the first transformation cell population comprisingPDX1-positive, NKX6.1-positive pancreatic progenitor cells with a secondcomposition comprising the PKC activator, a TGF-β signaling pathwayinhibitor, a thyroid hormone signaling pathway activator, and anepigenetic modifying compound, for one to two days, thereby obtaining asecond transformation cell population comprising NKX6.1-positive,ISL1-positive endocrine cells. In some cases, the method furthercomprises contacting the second transformation cell population with acomposition comprising a TGF-β signaling pathway inhibitor, a thyroidhormone signaling pathway activator, and an epigenetic modifyingcompound, thereby generating a population of cells comprising pancreaticendocrine cells.

In some cases, the population of cells comprising pancreatic endocrinecells generated according to the method provided herein comprises: atleast about 4% cells expressing somatostatin, at least about 15% cellsexpressing glucagon, at most about 35% cells expressing VMAT1, or atleast about 40% cells expressing C-peptide, as measured by flowcytometry. In some cases, the population of cells comprising pancreaticendocrine cells comprises: at least about 50% more cells expressingsomatostatin, at least about 50% more cells expressing glucagon, atleast about 20% fewer cells expressing VMAT1, or at least about 10% morecells expressing C-peptide, as compared to a corresponding population ofcells which is generated without contacting with the PKC activator. Insome cases, the population of cells comprising pancreatic endocrinecells comprises: at least about 100% more cells expressing somatostatin,at least about 200% more cells expressing glucagon, at least about 50%fewer cells expressing VMAT1, or at least about 20% more cellsexpressing C-peptide, as compared to a corresponding population of cellswhich is generated without contacting with the PKC activator.

In some aspects, the present disclosure provides for method thatcomprises contacting a plurality of PDX1-positive, NKX6.1-negativepancreatic progenitor cells with one or more of a ROCK inhibitor, agrowth factor from TGFβ superfamily, a growth factor from FGF family, aRA signaling pathway activator, and a SHH pathway inhibitor, therebygenerating a first population of cells. In some cases, the methodfurther comprises contacting the first population of cells with a PKCactivator and a γ-secretase inhibitor and one or more of a ROCKinhibitor, a growth factor from the TGFβ superfamily, a growth factorfrom the FGF family, a RA signaling pathway activator, and a SHH pathwayinhibitor, thereby generating a second population of cells. In somecases, the method further comprises contacting the second population ofcells with a PKC activator, a γ-secretase inhibitor and one or more of aTGF-β signaling pathway inhibitor, a growth factor from EGF family, a RAsignaling pathway activator, a SHH pathway inhibitor, a TH signalingpathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMPsignaling pathway inhibitor, and an epigenetic modifying compound,thereby generating a third population of cells. In some cases, themethod comprises: (a) contacting a plurality of PDX1-positive,NKX6.1-negative pancreatic progenitor cells with one or more of a ROCKinhibitor, a growth factor from TGFβ superfamily, a growth factor fromFGF family, a RA signaling pathway activator, and a SHH pathwayinhibitor, thereby generating a first population of cells; (b)contacting the first population of cells with a PKC activator and aγ-secretase inhibitor and one or more of a ROCK inhibitor, a growthfactor from the TGFβ superfamily, a growth factor from the FGF family, aRA signaling pathway activator, and a SHH pathway inhibitor, therebygenerating a second population of cells; and (c) contacting the secondpopulation of cells with a PKC activator, a γ-secretase inhibitor andone or more of a TGF-β signaling pathway inhibitor, a growth factor fromEGF family, a RA signaling pathway activator, a SHH pathway inhibitor, aTH signaling pathway activator, a protein kinase inhibitor, a ROCKinhibitor, a BMP signaling pathway inhibitor, and an epigeneticmodifying compound, thereby generating a third population of cells. Insome cases, the method further comprises: (d) contacting the thirdpopulation of cells with one or more of a serum albumin protein, vitaminC, a TGF-β signaling pathway inhibitor, a SHH pathway inhibitor, a THsignaling pathway activator, a protein kinase inhibitor, a ROCKinhibitor, a BMP signaling pathway inhibitor, and an epigeneticmodifying compound, thereby generating a fourth population of cells. Insome cases, step (d) comprises contacting the third population of cellswith a PKC activator.

In some aspects, the present disclosure provides a method thatcomprises: (a) contacting a plurality of PDX1-positive, NKX6.1-negativepancreatic progenitor cells with one or more of a ROCK inhibitor, agrowth factor from TGFβ superfamily, a growth factor from FGF family, aRA signaling pathway activator, and a SHH pathway inhibitor, therebygenerating a first population of cells; (b) contacting the firstpopulation of cells with a PKC activator and one or more of a ROCKinhibitor, a growth factor from the TGFβ superfamily, a growth factorfrom the FGF family, a RA signaling pathway activator, and a SHH pathwayinhibitor, thereby generating a second population of cells; wherein thePKC activator is a benzolactam-derivative; and (c) contacting the secondpopulation of cells with at he PKC activator, a γ-secretase inhibitor,and one or more of a TGF-β signaling pathway inhibitor, a growth factorfrom EGF family, a RA signaling pathway activator, a SHH pathwayinhibitor, a TH signaling pathway activator, a protein kinase inhibitor,a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigeneticmodifying compound, thereby generating a third population of cells. Insome cases, the benzolactam-derivative is TPPB. In some cases, the step(b) for generating the second population of cells comprises contactingthe first population of cells with a γ-secretase inhibitor. In somecases, the method further comprises (d) contacting the third populationof cells with one or more of a TGF-β signaling pathway inhibitor, a RAsignaling pathway activator, a TH signaling pathway activator, a proteinkinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor,and an epigenetic modifying compound, thereby generating a fourthpopulation of cells. In some cases, step (d) for generating the fourthpopulation of cells does not comprise contacting the third population ofcells with a PKC activator. In some cases, step (d) for generating thefourth population of cells does not comprise contacting the thirdpopulation of cells with a γ-secretase inhibitor. In some cases, step(d) for generating the fourth population of cells does not comprisecontacting the third population of cells with a SHH pathway inhibitor.In some cases, step (d) for generating the fourth population of cellsdoes not comprise contacting the third population of cells with a growthfactor from EGF family.

In some cases, the method further comprises: (e) contacting the fourthpopulation of cells with one or more of a serum albumin protein, vitaminC, a TGF-β signaling pathway inhibitor, a SHH pathway inhibitor, a THsignaling pathway activator, a protein kinase inhibitor, a ROCKinhibitor, a BMP signaling pathway inhibitor, and an epigeneticmodifying compound, thereby generating a fifth population of cells. Insome cases, step (e) comprises contacting the fourth population of cellswith a PKC activator.

In some aspects, the present disclosure provides a method that comprises(a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreaticprogenitor cells with one or more of a ROCK inhibitor, a growth factorfrom TGFβ superfamily, a growth factor from FGF family, a RA signalingpathway activator, and a SHH pathway inhibitor, thereby generating afirst population of cells; (b) contacting the first population of cellswith a PKC activator and one or more of a ROCK inhibitor, a growthfactor from the TGFβ superfamily, a growth factor from the FGF family, aRA signaling pathway activator, and a SHH pathway inhibitor, therebygenerating a second population of cells; (c) contacting the secondpopulation of cells with a PKC activator and one or more of aγ-secretase inhibitor, a TGF-β signaling pathway inhibitor, a growthfactor from EGF family, a RA signaling pathway activator, a SHH pathwayinhibitor, a TH signaling pathway activator, a protein kinase inhibitor,a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigeneticmodifying compound, thereby generating a third population of cells; (d)contacting the third population of cells with one or more of a TGF-βsignaling pathway inhibitor, a RA signaling pathway activator, a THsignaling pathway activator, a protein kinase inhibitor, a ROCKinhibitor, a BMP signaling pathway inhibitor, and an epigeneticmodifying compound, thereby generating a fourth population of cells; and(e) contacting the fourth population of cells with a PKC activator andone or more of a serum albumin protein, vitamin C, a TGF-β signalingpathway inhibitor, a SHH pathway inhibitor, a TH signaling pathwayactivator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signalingpathway inhibitor, and an epigenetic modifying compound, therebygenerating a fifth population of cells. In some cases, the step (d) ofthe method provided herein comprises contacting the fourth population ofcells with a serum albumin protein.

In some cases, step (a) for generating the first population of cells inthe method disclosed herein is performed over the course of about 1, 2,3, 4, 5 or 6 days. In some cases, step (a) for generating the firstpopulation of cells is performed over the course of 3-5 days, forinstance 3-4 days, 4-5 days, about 3 days, about 4 days, or about 5days. In some cases, the step (a) for generating the first population ofcells is performed over the course of 4 days. In some cases, step (b)for generating the second population of cells in the method disclosedherein is performed over the course of 1, 2, 3 or 4 days. In some cases,step (b) for generating the second population of cells is performed overthe course of 1-3 days, for instance, 1-2 days, 2-3 days, about 1 day,about 2 days, or about 3 days. In some cases, step (b) for generatingthe second population of cells is performed over the course of 2 days.In some cases, step (c) for generating the third population of cells inthe method disclosed herein is performed over the course of 1, 2, 3, or4 days. In some cases, step (c) for generating the third population ofcells is performed over the course of 1-3 days, for instance, 1-2 days,2-3 days, about 1 day, about 2 days, or about 3 days. In some cases,step (c) for generating the third population of cells is performed overthe course of 2 days. In some cases, step (d) for generating the fourthpopulation of cells in the method disclosed herein is performed over thecourse of 1, 2, 3, 4, 5, 6, or 7 days. In some cases, step (d) forgenerating the fourth population of cells is performed over the courseof 4-6 days, for instance 5-6 days, 4-5 days, about 4 days, about 5days, or about 6 days. In some cases, step (d) for generating the fourthpopulation of cells is performed over the course of 5 days. In somecases, step (e) for generating the fifth population of cells in themethod disclosed herein is performed over the course of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. In some cases, step (d) forgenerating the fourth population of cells is performed over the courseof 10-12 days, for instance, 10-11 days, 11-12 days, about 10 days,about 11 days, about 12 days.

In some cases, the second population of cells comprises PDX1-positiveand NKX6.1-positive cells. In some cases, the fourth population of cellscomprises PDX1-positive, NKX6.1-positive, ISL1-positive cells. In somecases, the fifth population of cells comprises cells that expressC-peptide and ISL1 but not VMAT1. In some cases, 30-90%, 30-80%, 30-70%,30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%,50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%,70-80%, or 80-90% of the cells in the fourth population of cells expressC-peptide and ISL1 but not VMAT1. In some cases, 40-60% of the cells inthe fourth population of cells express C-peptide and ISL1 but not VMAT1.In some cases, the fourth population of cells comprises cells thatexpress glucagon but not somatostatin. In some cases, 5-40%, 5-35%,5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%,10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%,20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35% or 35-40% of thecells in the fourth population of cells express glucagon but notsomatostatin. In some cases, 10-25% of the cells in the fourthpopulation of cells express somatostatin but not glucagon. In somecases, the fourth population of cells comprises cells that expresssomatostatin but not glucagon. In some cases, 3-20%, 3-15%, 3-12%,3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%,5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%,8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or15-20% of the cells in the fourth population of cells expresssomatostatin but not glucagon.

In some cases, the step of generating the first population of cells inthe method provided herein comprises contacting a plurality ofPDX1-positive, NKX6.1-negative pancreatic progenitor cells with a ROCKinhibitor, a growth factor from TGFβ superfamily, a growth factor fromFGF family, a RA signaling pathway activator, and a SHH pathwayinhibitor. In some cases, the step of generating the second populationof cells in the method provided herein comprises contacting the firstpopulation of cells with a ROCK inhibitor, a growth factor from the TGFβsuperfamily, a growth factor from the FGF family, a RA signaling pathwayactivator, and a SHH pathway inhibitor. In some cases, the step ofgenerating the third population of cells in the method provided hereincomprises contacting the second population of cells with agamma-secretase inhibitor, a TGF-β signaling pathway inhibitor, a growthfactor from EGF family, a RA signaling pathway activator, a SHH pathwayinhibitor, a TH signaling pathway activator, a protein kinase inhibitor,a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigeneticmodifying compound. In some cases, the step of generating the fourthpopulation of cells in the method provided herein comprises contactingthe third population of cells with serum albumin protein, a TGF-βsignaling pathway inhibitor, a SHH pathway inhibitor, a TH signalingpathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMPsignaling pathway inhibitor, and an epigenetic modifying compound. Insome cases, the ROCK inhibitor for use in the method provided herein isthiazovavin. In some cases, the growth factor from the TGFβ superfamilyfor use in the steps of generating the first population of cells and/orthe second population of cells in the method provided herein is activinA. In some cases, the growth factor from the FGF family for use in thesteps of generating the first population of cells and/or the secondpopulation of cells in the method provided herein is KGF. In some cases,the RA signaling pathway activator for use in the steps of generatingthe first population of cells, the second population of cells, and/orthe third population of cells in the method provided herein is retinoicacid. In some cases, the SHH pathway inhibitor for use in the steps ofgenerating the first population of cells, the second population ofcells, and/or the third population of cells in the method providedherein is Sant-1. In some cases, the PKC activator for use in the stepsof generating the second population of cells, the third population ofcells, and/or the fourth population of cells in the method providedherein is selected from the group consisting of: phorbol12,13-dibutyrate (PDBU), FR 236924, Prostratin, SC-9, and TPPB. In somecases, the PKC activator is PDBU. In some cases, the γ-secretaseinhibitor for use in the steps of generating the second population ofcells, and/or the third population of cells in the method providedherein is XXI. In some cases, the TGF-β signaling pathway inhibitor foruse in the steps of generating the third population of cells, and/or thefourth population of cells in the method provided herein is ALK5i. Insome cases, the growth factor from the EGF family for use in the stepsof generating the third population of cells in the method providedherein is betacellulin. In some cases, the TH signaling pathwayactivator for use in the steps of generating the third population ofcells and/or the fourth population of cells in the method providedherein is T3, GC-1 or a thyroid hormone derivative. In some cases, theprotein kinase inhibitor for use in the steps of generating the thirdpopulation of cells, and/or the fourth population of cells in the methodprovided herein is staurosporine. In some cases, the BMP signalingpathway inhibitor for use in the steps of generating the thirdpopulation of cells, and/or the fourth population of cells in the methodprovided herein is LDN193189 or DMH-1. In some cases, the epigeneticmodifying compound for use in the steps of generating the thirdpopulation of cells, and/or the fourth population of cells in the methodprovided herein is DZNep.

In some cases, the first time period during which the pancreaticprogenitor cells are treated with PKC activator is at least two days,three days, or four days. In some cases, the first time period is atmost four days, three days, or two days. In some cases, the first timeperiod is from two to four days. In some cases, the second time periodduring which the pancreatic progenitor cells are treated with PKCactivator is at least two days. In some cases, the second time period isat most four days. In some cases, the second time period is from two tofour days. In some cases, treatment of PKC activator as discussed hereinduring the transition between differentiation of PDX1-positive,NKX6.1-positive pancreatic progenitor cells and differentiation ofNKX6.1-positive, ISL1-positive endocrine cells is for at least two days,three days, or four days. In some cases, treatment of PKC activator asdiscussed herein during the transition between differentiation ofPDX1-positive, NKX6.1-positive pancreatic progenitor cells anddifferentiation of NKX6.1-positive, ISL1-positive endocrine cells is forat most two days, three days, or four days. In some cases, treatment ofPKC activator as discussed herein during the transition betweendifferentiation of PDX1-positive, NKX6.1-positive pancreatic progenitorcells and differentiation of NKX6.1-positive, ISL1-positive endocrinecells is for from two days to four days.

In some embodiments, a PKC activator is contacted to a population ofdifferentiating cells at two or more different time points during thedifferentiation process. In some embodiments, the PKC activator iscontacted to a population of cells, wherein the cells comprisePDX1-positive, NKX6.1-negative cells. In some embodiments, the PKCactivator is contacted to a population of cells, wherein the cellscomprise PDX1-positive, NKX6.1-positive cells. In some embodiments, thePKC activator is contacted to a population of cells, wherein the cellscomprise insulin-positive cells. In some embodiments, the PKC activatoris contacted to a population of cells at each of the followingdifferentiation stages: when the cells comprise PDX1-positive,NKX6.1-negative cells; when the cells comprise PDX1-positive,NKX6.1-positive cells; and when the cells comprise insulin-positivecells. In some embodiments, the same type of PKC activator (e.g., aphorbol ester or benzolactam-derivative) is administered to thedifferent population of cells at the two or more different time points.For example, in some embodiments, a phorbol ester (e.g., PDBU) isadministered to a cell population comprising PDX1-positive,NKX6.1-negative cells, and a phorbol ester (e.g., PDBU) is administeredto a cell population comprising PDX-positive, NKX6.1-positive cellsduring the same differentiation protocol. In some embodiments, one ormore different PKC activators (e.g., a phorbol ester and abenzolactam-derivative) are administered to the different population ofcells at the two or more different time points. For example, in someembodiments, a phorbol ester (e.g., PDBU) is administered to a cellpopulation comprising PDX1-positive, NKX6.1-negative cells, and abenzolactam derivative (e.g., TPPB) is administered to a cell populationcomprising PDX-positive, NKX6.1-positive cells during the samedifferentiation protocol.

In some cases, non-limiting examples of the PKC activator of the methoddescribed herein include phorbol 12,13-dibutyrate (PDBU), FR 236924,Prostratin, SC-9, and TPPB. In some cases, the PKC activator comprisesPDBU. In some cases, the PKC activator comprises TPPB. In some cases,the PKC activator is contacted to the population of cells comprisingPDX1-positive, NKX6.1-positive pancreatic progenitor cells at aconcentration from 50 nM to 2000 nM, from 75 nM to 1500 nM, from 100 nMto 1000 nM, from 200 nM to 750 nM, or from 400 nM to 600 nM. In somecases, the PKC activator is at a concentration from 100 nM to 1000 nM.In some cases, the PKC activator is at a concentration at least about100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM,or 1000 nM. In some cases, the PKC activator is at a concentration atmost about 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800nM, 900 nM, or 1000 nM. In some cases, the PKC activator is at aconcentration about 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700nM, 800 nM, 900 nM, or 1000 nM. In some cases, the PKC activator is at aconcentration about 500 nM.

In some cases, non-limiting examples of the gamma secretase inhibitorused in the methods described herein include XXI and DAPT. In somecases, the gamma secretase inhibitor comprises XXI. In some cases, thegamma secretase inhibitor is contacted to the population of cellscomprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells ata concentration from 0.2 μM to 20 μM, from 0.3 μM to 15 μM, or from 0.5μM to 10 μM, from 1μM to 5 μM, or from 1.5 μM to 2.5 μM. In some cases,the gamma secretase inhibitor is at a concentration about 0.5 μM, 0.75μM, 1 μM, 1.25 μM, 1.5 μM, 1.75 μM, 2 μM, 2.25 μM, 2.5 μM, 3 μM, 4 μM, 5μM, 7.5 μM, 10 μM, 15 μM, or 20 μM. In some cases, the gamma secretaseinhibitor is at a concentration at least about 0.5 μM, 0.75 μM, 1 μM,1.25 μM, 1.5 μM, 1.75 μM, 2 μM, 2.25 μM, 2.5 μM, 3 μM, 4 μM, or 5 μM. Insome cases, the gamma secretase inhibitor is at a concentration at mostabout 1 μM, 1.25 μM, 1.5 μM, 1.75 μM, 2 μM, 2.25 μM, 2.5 μM, 3 μM, 4 μM,5 μM, 7.5 μM, 10 μM, 15 μM, or 20 μM.

Cell Compositions

In some aspects, provided herein are cell compositions that include SC-βcells, SC-α cells, SC-δ cells, and SC-EC cells. In some cases, the cellcompositions provided herein have desirable amount (e.g., percentage) ofSC-β cells, SC-α cells, and SC-δ cells, and limited amount of SC-ECcells. In some cases, the cell constituent of the cell compositionsresembles a native pancreatic islet.

In some cases, the SC-β cells of the disclosure share manycharacteristic features of β cells which are important for normal β cellfunction. In some embodiments, the SC-β cell exhibits a glucosestimulated insulin secretion (GSIS) response in vitro. In someembodiments, the SC-β cell exhibits a GSIS response in vivo. In someembodiments, the SC-β cell exhibits in vitro and in vivo GSIS responses.In some embodiments, the GSIS responses resemble the GSIS responses ofan endogenous mature pancreatic β cell. In some embodiments, the SC-βcell exhibits a GSIS response to at least one glucose challenge. In someembodiments, the SC-β cell exhibits a GSIS response to at least twosequential glucose challenges. In some embodiments, the SC-β cellexhibits a GSIS response to at least three sequential glucosechallenges. In some embodiments, the GSIS responses resemble the GSISresponse of endogenous human islets to multiple glucose challenges. Insome embodiments, the GSIS response is observed immediately upontransplanting the cell into a human or animal. In some embodiments, theGSIS response is observed within approximately 24 hours of transplantingthe cell into a human or animal. In some embodiments, the GSIS responseis observed within approximately one week of transplanting the cell intoa human or animal. In some embodiments, the GSIS response is observedwithin approximately two weeks of transplanting the cell into a human oranimal. In some embodiments, the stimulation index of the cell ascharacterized by the ratio of insulin secreted in response to highglucose concentrations compared to low glucose concentrations is similarto the stimulation index of an endogenous mature pancreatic β cell. Insome embodiments, the SC-β cell exhibits a stimulation index of greaterthan 1. In some embodiments, the SC-β cell exhibits a stimulation indexof greater than or equal to 1. In some embodiments, the SC-β cellexhibits a stimulation index of greater than 1.1. In some embodiments,the SC-β cell exhibits a stimulation index of greater than or equal to1.1. In some embodiments, the SC-β cell exhibits a stimulation index ofgreater than 2. In some embodiments, the SC-β cell exhibits astimulation index of greater than or equal to 1. In some embodiments,the SC-β cell exhibits a stimulation index of at least 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 orgreater.

In some embodiments, the disclosure provides for an in vitrocomposition, comprising PDX1-positive, NKX6.1-positive pancreaticprogenitor cells; a PKC activator; and a γ-secretase inhibitor. In someembodiments, the disclosure provides for an in vitro composition,comprising NKX6.1-positive, ISL1-positive endocrine cells; a PKCactivator; and a γ-secretase inhibitor. In some embodiments, thedisclosure provides for an in vitro composition, comprisingPDX1-positive, NKX6.1-positive pancreatic progenitor cells;NKX6.1-positive, ISL1-positive endocrine cells; a PKC activator; and aγ-secretase inhibitor. In some embodiments, the PKC activator isselected from the group consisting of: phorbol 12,13-dibutyrate (PDBU),FR 236924, Prostratin, SC-9, and TPPB. In some embodiments, theγ-secretase inhibitor is DAPT or XXI.

In some aspects, the present disclosure provides an in vitro compositionthat comprises PDX1-positive, NKX6.1-negative pancreatic progenitorcells; PDX1-positive, NKX6.1-positive pancreatic progenitor cells; a PKCactivator; and a γ-secretase inhibitor. In some embodiments, at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the cells in thecomposition are PDX1-positive, NKX6.1-positive pancreatic progenitorcells. In some embodiments, less than 90%, 80%, 70%, 60%, 50%, 40%, 30%,20%, or 10% of the cells in the composition are PDX1-positive,NKX6.1-negative pancreatic progenitor cells. In some embodiments, thePKC activator is selected from the group consisting of: phorbol12,13-dibutyrate (PDBU), FR 236924, Prostratin, SC-9, and TPPB. In someembodiments, the γ-secretase inhibitor is DAPT or XXI. In someembodiments, the composition further comprises a growth factor from theFGF family. In some embodiments, the growth factor from the FGF familyis KGF. In some embodiments, the composition further comprises a growthfactor of the TGFβ superfamily. In some embodiments, the growth factorof the TGFβ superfamily is activin A.

In some aspects, the present disclosure provides an in vitro compositioncomprising PDX1-positive cells, a γ-secretase inhibitor, and one or bothof a growth factor from the TGFβ superfamily and a growth factor fromthe FGF family. In some embodiments, the composition of cells comprisesPDX1-positive, NKX6.1-negative cells. In some embodiments, thecomposition of cells comprises PDX1-positive, NKX6.1-positive cells. Insome embodiments, the composition further comprises any one of orcombination of a PKC activator, a growth factor from the FGF family, aROCK inhibitor, a growth factor from the TGFβ superfamily, a sonichedgehog pathway inhibitor, and a retinoic acid signaling pathwayactivator.

In some aspects, the present disclosure provides an in vitro compositioncomprising PDX1-positive, NKX6.1-negative pancreatic progenitor cells;PDX1-positive, NKX6.1-positive pancreatic progenitor cells; and aγ-secretase inhibitor. In some embodiments, the γ-secretase inhibitor isXXI. In some embodiments, the γ-secretase inhibitor is DAPT.

In some embodiments, at least 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least90% of the cells in the composition are PDX1-positive, NKX6.1-positivepancreatic progenitor cells. In some embodiments, less than 90%, lessthan 80%, less than 70%, less than 60%, less than 50%, less than 40%,less than 30%, less than 20%, or less than 10% of the cells in thecomposition are PDX1-positive, NKX6.1-negative pancreatic progenitorcells.

In some embodiments, the composition further comprises a growth factorfrom the FGF family. In some embodiments, the composition furthercomprises a sonic hedgehog pathway inhibitor. In some embodiments, thecomposition further comprises a ROCK inhibitor. In some embodiments, thecomposition further comprises a growth factor from the TGFβ superfamily.In some embodiments, the composition further comprises a retinoic acidsignaling pathway activator. In some embodiments, the compositionfurther comprises a PKC activator.

In some embodiments, the composition further comprises two or more(e.g., any two, any three, any four, any five, or any six) of a PKCactivator, a growth factor from the FGF family, a ROCK inhibitor, agrowth factor from the TGFβ superfamily, a sonic hedgehog pathwayinhibitor, and a retinoic acid signaling pathway activator. In someembodiments of the composition, the growth factor from the FGF family isKGF. In some embodiments, the sonic hedgehog pathway inhibitor isSANT-1. In some embodiments, the ROCK inhibitor is thiazovivin. In someembodiments, the growth factor from the TGFβ superfamily is activin A.In some embodiments, the retinoic acid signaling pathway activator isretinoic acid. In some embodiments, the PKC activator is PDBU.

In some aspects, the present disclosure provides a population of invitro differentiated cells comprising NKX6.1-positive, ISL1-positivecells and NKX6.1-negative, ISL1-positive cells. In some embodiments, thepopulation comprises more NKX6.1-negative, ISL1-positive cells thanNKX6.1-positive, ISL1-positive cells. In some embodiments, at least 73%of the cells in the population are ISL1-positive cells. In someembodiments, at least 40% of the cells in the population areNKX6.1-negative, ISL1-positive cells. In some embodiments, less than 12%of the cells in the population are NKX6.1-negative, ISL1-negative cells.

In some aspects, the present disclosure provides a population of invitro differentiated cells comprising NKX6.1-positive, ISL1-positivecells and NKX6.1-negative, ISL1-positive cells, where less than 12% ofthe cells (e.g., about 11%, about 10%, about 9%, about 8%, about 7%,about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less) inthe population are NKX6.1-negative, ISL1-negative cells. In someembodiments, less than 10%, less than 8%, less than 6%, less than 4%,1-11%, 2-10%, 2-12%, 4-12%, 6-12%, 8-12%, 2-8%, 4-8%, 3-6% or 3-5% ofthe cells in the population are NKX6.1-negative, ISL1-negative cells. Insome embodiments, 2-12%, 4-12%, 6-12%, 8-12%, 2-8%, 4-8%, 3-6% or 3-5%of the cells in the population are NKX6.1-negative, ISL1-negative cells.

In some embodiments, at least 60%, at least 65%, at least 70%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, about 85-95%, or about 90-95% of the cells in the population areISL1-positive cells. In some embodiments, 50-90%, 50-85%, 50-80%,50-75%, 50-70%, 50-60%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 65-90%,65-85%, 65-80%, 65-75%, 65-70%, 70-90%, 70-85%, 70-80%, 70-75%, 75-90%,75-85%, 75-80%, 80-90%, 80-85%, or 85-90% of the cells in the populationare ISL1-positive cells. In some embodiments, at least 74%, at least75%, at least 80%, at least 85%, at least 90%, about 85-95%, or about90-95% of the cells in the population are ISL1-positive cells. In someembodiments, about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or about 99% of the cells in the population are ISL1-positivecells.

In some embodiments, the population comprises more NKX6.1-negative,ISL1-positive cells than NKX6.1-positive, ISL1-positive cells. In someembodiments, at least 40% of the cells in the population areNKX6.1-negative, ISL1-positive cells. In some embodiments, at least 45%,at least 50%, about 40-50%, about 45-55%, or about 50-55% of the cellsin the population are NKX6.1-negative, ISL1-positive cells. In someembodiments, about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, or about 55% of the cells in the population areNKX6.1-negative, ISL1-positive cells.

In some aspects, the present disclosure provides an in vitro compositionthat comprises PDX1-positive, NKX6.1-positive pancreatic progenitorcells; NKX6.1-positive, ISL1-positive endocrine cells; and a PKCactivator; wherein the PKC activator is a benzolactam derivative. Insome cases, the benzolactam is TPPB. In some cases, the compositionfurther comprises a γ-secretase inhibitor. The γ-secretase inhibitor canbe XXI.

In some cases, the composition provided herein comprises adifferentiation factor selected from the group consisting of: a TGF-βsignaling pathway inhibitor, a thyroid hormone signaling pathwayactivator, an epigenetic modifying compound, a growth factor from EGFfamily, a RA signaling pathway activator, a SHH pathway inhibitor, aprotein kinase inhibitor, a ROCK inhibitor, and a BMP signaling pathwayinhibitor. In some cases, the composition also comprises serum albuminprotein.

In some cases, the composition provided herein comprises serum albuminprotein, a TGF-β signaling pathway inhibitor, a thyroid hormonesignaling pathway activator, an epigenetic modifying compound, a SHHpathway inhibitor, a protein kinase inhibitor, a ROCK inhibitor, and aBMP signaling pathway inhibitor.

In some cases, the ROCK inhibitor is thiazovavin. In some cases, the RAsignaling pathway activator is retinoic acid. In some cases, the SHHpathway inhibitor is Sant-1. In some cases, the TGF-β signaling pathwayinhibitor is ALK5i. In some cases, the growth factor from the EGF familyis betacellulin. In some cases, the thyroid hormone signaling pathwayactivator is T3, GC-1 or a thyroid hormone derivative. In some cases,the protein kinase inhibitor is staurosporine. In some cases, the BMPsignaling pathway inhibitor is LDN193189 or DMH-1. In some cases, theepigenetic modifying compound is DZNep.

In some cases, the cell compositions of the present disclosure have atleast about 35% cells expressing C-peptide and not expressing VMAT1, asmeasured by flow cytometry. In some cases, the expression of C-peptideand absence of VMAT1 in a cell of the cell compositions suggest that thecell is a SC-β cell. In some cases, the cell compositions have at leastabout 30%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, or 50% cells expressing C-peptide and notexpressing VMAT1, as measured by flow cytometry. In some cases, the cellcompositions have about 30%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, or 60% cells expressing C-peptide and notexpressing VMAT1, as measured by flow cytometry. In some cases, the cellcompositions have about 30% to about 60%, about 35% to about 55%, about40% to about 50% cells expressing C-peptide and not expressing VMAT1, asmeasured by flow cytometry.

In some cases, the cell compositions of the present disclosure have atmost about 35% cells expressing VMAT1, as measured by flow cytometry. Insome cases, the cell compositions of the present disclosure have at mostabout 35% cells expressing VMAT1 and not expressing C-peptide, asmeasured by flow cytometry. In some cases, the expression of VMAT1 andabsence of C-peptide in a cell of the cell compositions suggest that thecell is a SC-EC cell. In some cases, the cell compositions have at mostabout 35%, 32%, 31%, 30%, 28%, 25%, 24%, 23%, 22%, 21%, or 20% cellsexpressing VMAT1 and not expressing C-peptide, as measured by flowcytometry. In some cases, the cell compositions have about 35%, 32%,31%, 30%, 28%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, or 15%cells expressing VMAT1 and not expressing C-peptide, as measured by flowcytometry. In some cases, the cell compositions have about 15% to about30%, about 16% to 25%, about 17% to about 22%, about 18% to about 20%cells expressing VMAT1 and not expressing C-peptide, as measured by flowcytometry.

In some cases, the cell composition include at least about 20% cellsexpressing glucagon, as measured by flow cytometry. In some cases, thecell composition include at least about 15% cells expressing glucagonand not expressing somatostatin, as measured by flow cytometry. In somecases, the expression of glucagon and not expressing somatostatin in acell of the cell composition suggest that the cell is a SC-α cell. Insome cases, the cell composition include at least about 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, or 22% cells expressing glucagon andnot expressing somatostatin, as measured by flow cytometry. In somecases, the cell composition include about 10% to about 30%, about 12% toabout 25%, about 13% to about 22%, about 15% to about 20%, or about 16%to about 18% cells expressing glucagon and not expressing somatostatin,as measured by flow cytometry. In some cases, the cell compositioninclude about 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, or 22%cells expressing glucagon and not expressing somatostatin, as measuredby flow cytometry.

In some cases, the cell composition include at least about 4% cellsexpressing somatostatin and not expressing glucagon, as measured by flowcytometry. In some cases, the expression of glucagon and not expressingsomatostatin in a cell of the cell composition suggest that the cell isa SC-δ cell. In some cases, the cell composition include at least about2%, 3%, 4%, 5%, 6%, 7%, or 8% cells expressing somatostatin and notexpressing glucagon, as measured by flow cytometry. In some cases, thecell composition include about 1% to about 9%, about 2% to about 8%,about 3% to about 7%, or about 4% to about 6% cells expressingsomatostatin and not expressing glucagon, as measured by flow cytometry.In some cases, the cell composition include about 2%, 3%, 4%, 5%, 6%,7%, or 8% cells expressing somatostatin and not expressing glucagon, asmeasured by flow cytometry.

In some cases, the cell composition have at least about 35% cellsexpressing C-peptide and not expressing VMAT1, at most about 30% cellsexpressing VMAT1, and at least about 20% cells expressing glucagon, asmeasured by flow cytometry. In some cases, the cell composition have atleast about 35% cells expressing C-peptide and not expressing VMAT1, atmost about 30% cells expressing VMAT1, at least about 20% cellsexpressing glucagon, and at least 4% cells expressing somatostatin andnot expressing glucagon, as measured by flow cytometry.

In some cases, the cell composition provided herein include (a) at leastabout 35% cells expressing C-peptide and not expressing VMAT1; and (b)at least about 10% cells expressing somatostatin, as measured by flowcytometry. In some cases, there are at least about 15% cells expressingsomatostatin in the cell composition, as measured by flow cytometry.

In some cases, provided herein is a composition comprising a populationof cells, wherein: (a) 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%,40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%,60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-80%, or 80-90% of thecells in the population of cells express C-peptide and ISL1 but notVMAT1; (b) 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%,10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%,15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%,30-35% or 35-40% of the cells in the population of cells expressglucagon but not somatostatin; and/or (c) 3-20%, 3-15%, 3-12%, 3-10%,3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%,5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%,8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or 15-20% ofthe cells in the population of cells express somatostatin but notglucagon.

In some cases, provided herein is a composition comprising a populationof cells, wherein: (a) 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%,40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%,60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-80%, or 80-90% of thecells in the population of cells express C-peptide and ISL1 but notVMAT1; (b) 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%,10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%,15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%,30-35% or 35-40% of the cells in the population of cells expressglucagon but not somatostatin; and (c) 3-20%, 3-15%, 3-12%, 3-10%, 3-8%,3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%,5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%,8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or 15-20% ofthe cells in the population of cells express somatostatin but notglucagon.

In some cases, in the population of cells provided herein, 40-60% of thecells express C-peptide and ISL1 but not VMAT1; 10-25%, of the cellsexpress glucagon but not somatostatin; and 4-10% of the cells expresssomatostatin but not glucagon. In some cases, less than 25%, less than20%, less than 18%, less than 15%, less than 12%, or less than 10% ofthe cells in the population of cells provided herein express VMAT1 butnot C-peptide.

Still other embodiments of the present disclosure relate tocompositions, such as isolated cell populations or cell cultures,comprising mixtures of SC-β cells and insulin-positive endocrine cellsor precursors thereof from which they were differentiated from. Forexample, cell cultures or cell populations comprising at least about 5SC-β cells for about every 95 insulin-positive endocrine cells orprecursors thereof can be produced. In other embodiments, cell culturesor cell populations comprising at least about 95 SC-β cells for aboutevery 5 insulin-positive endocrine cells or precursors thereof can beproduced. Additionally, cell cultures or cell populations comprisingother ratios of SC-β cells to insulin-positive endocrine cells orprecursors thereof are contemplated. For example, compositionscomprising at least about one SC-β cell for about every 1,000,000, or atleast 100,000 cells, or at least 10,000 cells, or at least 1000 cells or500, or at least 250 or at least 100 or at least 10 insulin-positiveendocrine cells or precursors thereof can be produced.

In some cases, cell populations or cell clusters disclosed herein areunsorted, e.g., isolated cell populations or cell clusters that have notbeen through cell sorting process. In some embodiments, the cell clusterdisclosed herein can refer to a cell cluster formed by self-aggregationof cells cultured in a given environment, for instance, in a 3Dsuspension culture. In some embodiments, cell clusters disclosed hereinare intermediate cell clusters formed during the differentiation processas described herein. In some cases, the intermediate cell clusters,e.g., cell clusters comprising PDX1-positive, NKX6.1-negative pancreaticprogenitor cells (e.g., Stage 3 cell clusters) or cell clusterscomprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells(e.g., Stage 4 cell clusters), are not subjected to cell sorting. Insome case, cell populations going through cell sorting may not be ableto form the intermediate cell clusters disclosed herein. For instance,PDX1-positive pancreatic progenitor cells, after going through cellsorting, may not be able to form α cell cluster as disclosed herein.

Cell sorting as described herein can refer to a process of isolating agroup of cells from a plurality of cells by relying on differences incell size, shape (morphology), surface protein expression, endogenoussignal protein expression, or any combination thereof. In some cases,cell sorting comprises subjecting the cells to flow cytometry. Flowcytometry can be a laser- or impedance-based, biophysical technology.During flow cytometry, one can suspend cells in a stream of fluid andpass them through an electronic detection apparatus. In one type of flowcytometry, fluorescent-activated cell sorting (FACS), based on one ormore parameters of the cells' optical properties (e.g., emission wavelength upon laser excitation), one can physically separate and therebypurify cells of interest using flow cytometry. As described herein, anunsorted cell cluster can be cell cluster that formed by a plurality ofcells that have not been subject to an active cell sorting process,e.g., flow cytometry. In some cases, flow cytometry as discussed hereincan be based on one or more signal peptides expressed in the cells. Forexample, a cell cluster can comprise cells that express a signal peptide(e.g., a fluorescent protein, e.g., green fluorescent protein (GFP) ortdTomato). In some cases, the signal peptide is expressed as anindicator of insulin expression in the cells. For instance, a cellcluster can comprise cell harboring an exogenous nucleic acid sequencecoding for GFP under the control of an insulin promoter. The insulinpromoter can be an endogenous or exogenous promoter. In some cases, theexpression of GFP in these cells can be indicative of insulin expressionin the cells. The GFP signal can thus be a marker of a pancreatic βcell. In some cases, cell sorting as described herein can comprisemagnetic-activated flow cytometry, where magnetic antibody or otherligand is used to label cells of different types, and the differences inmagnetic properties can be used for cell sorting.

The percentage of cells expressing one or more particular markers, likePDX1, NKX6.1, insulin, NGN3, or CHGA, described herein can be thepercentage value detected using techniques like flow cytometry assay. Insome cases, during a flow cytometry assay, cell population or cellcluster discussed herein are dispersed into single-cell suspension byincubation in digesting enzyme like trypsin or TrypLE™ Express.Dispersed cell can be washed in suitable buffer like PBS, centrifugedand then re-suspended in fixation buffer like 4%PFA. Incubation withprimary antibodies against the cell markers of interest can then beconducted, which can be followed by incubation with the secondaryantibodies. After antibody incubation, the cells can be washed and thesubject to segregation by flow cytometry. Techniques other than flowcytometry can also be used to characterize the cells described herein,e.g., determine the cell percentages. Non-limiting examples of cellcharacterization methods include gene sequencing, microscopic techniques(fluorescence microscopy, atomic force microscopy), karyotyping,isoenzyme analysis, DNA properties, and viral susceptibility.

In some aspects, the disclosure relates to a composition comprising apopulation of glucose-responsive insulin secreting cells, wherein thecells secrete a higher amount of insulin upon induction with KCl (e.g.,about 20 to about 50 mM, e.g., about 30 mM) as compared to the amount ofinsulin secreted upon induction with glucose. In some embodiments, thepopulation of glucose-responsive insulin secreting cells secrete atleast 1.5 times, 2 times, 2.5 times, 3 times higher amount of insulinupon induction with KCl as compared to the amount of insulin secretedupon induction with glucose.

In some aspects, the disclosure relates to a composition comprising apopulation of glucose-responsive insulin secreting cells, wherein thecells secrete a higher amount of insulin upon induction with KCl and/orglucose, in the presence of a signaling factor as compared to comparablecells in the absence of the signaling factor. In some embodiments, thecells secrete higher amount of insulin in the presence of high glucose,but not in the presence of low glucose. In some embodiments, the highglucose concentration is about 10-20 mM. In some embodiments, the lowglucose concentration is about 2-5 mM.

In some aspects, the disclosure relates to a composition comprising apopulation of differentiated pancreatic progenitor cells, wherein thepopulation comprises at least 60% pancreatic β cells as determined byflow cytometry. In some embodiments, the population comprises at least65%, 70%, 75%, 80%, 85%, or 90% pancreatic β cells. In some embodiments,the population comprises a higher percentage of pancreatic β cells uponbeing contacted with a predetermined basal medium component as comparedto a comparable population not contacted with the basal mediumcomponent.

The in vitro-matured, SC-β cell (e.g., pancreatic β cells) generatedaccording to the disclosed methods described herein demonstrate manyadvantages, for example, they perform glucose stimulated insulinsecretion in vitro, resemble human islet β cells by gene expression andultrastructure, secrete human insulin and ameliorate hyperglycemia whentransplanted into mice, provide a new platform for cell therapy (e.g.,transplantation into a subject in need of additional and/or functional βcells), drug screening (e.g., for insulin production/secretion,survival, dedifferentiation, etc.), research (e.g., determining thedifferences in function between normal and diabetic β cell), and tissueengineering (e.g., using the SC-β cells as the first cell type inreconstructing an islet).

Stem Cells and Reprogramming

Provided herein is use of stem cells for producing SC-β cells (e.g.,mature pancreatic β cells or β-like cells) or precursors thereof. In anembodiment, germ cells may be used in place of, or with, the stem cellsto provide at least one SC-β cell, using similar protocols as describedin U.S. Patent Application Publication No. US20150240212 andUS20150218522, each of which is herein incorporated by reference in itsentirety. Suitable germ cells can be prepared, for example, fromprimordial germ cells present in human fetal material taken about 8-11weeks after the last menstrual period. Illustrative germ cellpreparation methods are described, for example, in Shamblott et al.,Proc. Natl. Acad. Sci. USA 95:13726, 1998 and U.S. Pat. No. 6,090,622.

Provided herein are compositions and methods of generating SC-β cells(e.g., pancreatic β cells), as well as pancreatic α cells, and/orpancreatic δ cells. In some embodiments, the disclosure provides formethods of generating cell populations that are enriched for pancreaticα cells. In some embodiments, the disclosure provides for methods ofgenerating cell populations that are enriched for pancreatic δ cells.

Generally, the at least one SC-β cell or precursor thereof, e.g.,pancreatic progenitors produced according to the methods disclosedherein can comprise a mixture or combination of different cells, e.g.,for example a mixture of cells such as primitive gut tube cells,PDX1-positive pancreatic progenitors, PDX1-positive, NKX6.1-positivepancreatic progenitors, Ngn3-positive endocrine progenitor cells,insulin-positive endocrine cell (e.g., NKX6.1-positive, ISL1-positivecells, or β-like cells), and/or other pluripotent or stem cells.

The at least one pancreatic α, β and/or δ cell or precursor thereof canbe produced according to any suitable culturing protocol todifferentiate a stem cell or pluripotent cell to a desired stage ofdifferentiation. In some embodiments, the at least one pancreatic α, βand/or δ cell or the precursor thereof are produced by culturing atleast one pluripotent cell for a period of time and under conditionssuitable for the at least one pluripotent cell to differentiate into theat least one pancreatic α, β and/or δ cell or the precursor thereof.

In some embodiments, the at least one pancreatic α, β and/or δ cell orprecursor thereof is a substantially pure population of pancreatic α, βand/or δ cells or precursors thereof. In some embodiments, a populationof pancreatic α, β and/or δ cells or precursors thereof comprises amixture of pluripotent cells or differentiated cells. In someembodiments, a population pancreatic α, β and/or δ cells or precursorsthereof are substantially free or devoid of embryonic stem cells orpluripotent cells or iPS cells.

In some embodiments, a somatic cell, e.g., fibroblast can be isolatedfrom a subject, for example as a tissue biopsy, such as, for example, askin biopsy, and reprogrammed into an induced pluripotent stem cell forfurther differentiation to produce the at least one pancreatic α, βand/or δ cell or precursor thereof for use in the compositions andmethods described herein. In some embodiments, a somatic cell, e.g.,fibroblast is maintained in culture by methods known by one of ordinaryskill in the art, and in some embodiments, propagated prior to beingconverted into pancreatic α, β and/or δ cells by the methods asdisclosed herein.

In some embodiments, the at least one pancreatic α, β and/or δ cell orprecursor thereof are maintained in culture by methods known by one ofordinary skills in the art, and in some embodiments, propagated prior tobeing converted into pancreatic α, β and/or δ cells by the methods asdisclosed herein.

Further, at least one pancreatic α, β and/or δ cell or precursorthereof, e.g., pancreatic progenitor can be from any mammalian species,with non-limiting examples including a murine, bovine, simian, porcine,equine, ovine, or human cell. For clarity and simplicity, thedescription of the methods herein refers to a mammalian at least onepancreatic α, β and/or δ cell or precursor thereof but it should beunderstood that all of the methods described herein can be readilyapplied to other cell types of at least one pancreatic α, β and/or δcell or precursor thereof. In some embodiments, the at least onepancreatic α, β and/or δ cell or precursor thereof is derived from ahuman individual.

Stem Cells

Embodiments of the present disclosure are related to use of stem cellsfor generation of pancreatic α, β and/or δ cells or precursors thereof.The term “stem cell” as used herein can refer to a cell (e.g., plantstem cell, vertebrate stem cell) that has the ability both to self-renewand to generate a differentiated cell type (Morrison et al., (1997) Cell88:287-298). In the context of cell ontogeny, the adjective“differentiated”, or “differentiating” is a relative term. A“differentiated cell” can be α cell that has progressed further down thedevelopmental pathway than the cell it is being compared with. Thus,pluripotent stem cells can differentiate into lineage-restrictedprogenitor cells (e.g., mesodermal stem cells), which in turn candifferentiate into cells that are further restricted (e.g., neuronprogenitors), which can differentiate into end-stage cells (e.g.,terminally differentiated cells, e.g., neurons, cardiomyocytes, etc.),which play a characteristic role in a certain tissue type, and can orcannot retain the capacity to proliferate further. Stem cells can becharacterized by both the presence of specific markers (e.g., proteins,RNAs, etc.) and the absence of specific markers. Stem cells can also beidentified by functional assays both in vitro and in vivo, particularlyassays relating to the ability of stem cells to give rise to multipledifferentiated progeny. In an embodiment, the host cell is an adult stemcell, a somatic stem cell, a non-embryonic stem cell, an embryonic stemcell, hematopoietic stem cell, an include pluripotent stem cells, and atrophoblast stem cell.

Stem cells of interest, e.g., that can be used in the method providedherein, can include pluripotent stem cells (PSCs). The term “pluripotentstem cell” or “PSC” as used herein can refer to a stem cell capable ofproducing all cell types of the organism. Therefore, a PSC can give riseto cells of all germ layers of the organism (e.g., the endoderm,mesoderm, and ectoderm of a vertebrate). Pluripotent cells can becapable of forming teratomas and of contributing to ectoderm, mesoderm,or endoderm tissues in a living organism. Pluripotent stem cells ofplants can be capable of giving rise to all cell types of the plant(e.g., cells of the root, stem, leaves, etc.).

Embodiments of the present disclosure are related to use of PSCs forgeneration of pancreatic α, β and/or δ cells or precursors thereof. PSCsof animals can be derived in a number of different ways. For example,embryonic stem cells (ESCs) can be derived from the inner cell mass ofan embryo (Thomson et. al, Science. 1998 Nov. 6; 282(5391):1145-7)whereas induced pluripotent stem cells (iPSCs) can be derived fromsomatic cells (Takahashi et. al, Cell. 2007 Nov. 30; 131(5):861-72;Takahashi et. al, Nat Protoc. 2007; 2(12):3081-9; Yu et. al, Science.2007 Dec. 21; 318(5858):1917-20. Epub 2007 Nov. 20). Because the termPSC can refer to pluripotent stem cells regardless of their derivation,the term PSC can encompass the terms ESC and iPSC, as well as the termembryonic germ stem cells (EGSC), which are another example of a PSC.PSCs can be in the form of an established cell line, they can beobtained directly from primary embryonic tissue, or they can be derivedfrom a somatic cell.

Embodiments of the present disclosure are related to use of ESCs forgeneration of pancreatic β cells or precursors thereof. By “embryonicstem cell” (ESC) can be meant a PSC that is isolated from an embryo,typically from the inner cell mass of the blastocyst. ESC lines arelisted in the NIH Human Embryonic Stem Cell Registry, e.g. hESBGN-01,hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3,HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMediHospital-Seoul National University); HSF-1, HSF-6 (University ofCalifornia at San Francisco); and H1, H7, H9, H13, H14 (Wisconsin AlumniResearch Foundation (WiCell Research Institute)). Stem cells of interestalso include embryonic stem cells from other primates, such as Rhesusstem cells and marmoset stem cells. The stem cells can be obtained fromany mammalian species, e.g. human, equine, bovine, porcine, canine,feline, rodent, e.g. mice, rats, hamster, primate, etc. (Thomson et al.(1998) Science 282:1145; Thomson et al. (1995) Proc. Natl. Acad. Sci USA92:7844; Thomson et al. (1996) Biol. Reprod. 55:254; Shamblott et al.,Proc. Natl. Acad. Sci. USA 95:13726, 1998). In culture, ESCs can grow asflat colonies with large nucleo-cytoplasmic ratios, defined borders andprominent nucleoli. In addition, ESCs can express SSEA-3, SSEA-4,TRA-1-60, TRA-1-81, and Alkaline Phosphatase, but not SSEA-1. Examplesof methods of generating and characterizing ESCs can be found in, forexample, U.S. Pat. No. 7,029,913, U.S. Pat. No. 5,843,780, and U.S. Pat.No. 6,200,806, each of which is incorporated herein by its entirety.Methods for proliferating hESCs in the undifferentiated form aredescribed in WO 99/20741, WO 01/51616, and WO 03/020920, each of whichis incorporated herein by its entirety.

By “embryonic germ stem cell” (EGSC) or “embryonic germ cell” or “EGcell”, it can be meant a PSC that is derived from germ cells and/or germcell progenitors, e.g. primordial germ cells, e.g. those that can becomesperm and eggs. Embryonic germ cells (EG cells) are thought to haveproperties similar to embryonic stem cells as described above. Examplesof methods of generating and characterizing EG cells may be found in,for example, U.S. Pat. No. 7,153,684; Matsui, Y., et al., (1992) Cell70:841; Shamblott, M., et al. (2001) Proc. Natl. Acad. Sci. USA 98: 113;Shamblott, M., et al. (1998) Proc. Natl. Acad. Sci. USA, 95:13726; andKoshimizu, U., et al. (1996) Development, 122:1235, each of which areincorporated herein by its entirety.

Embodiments of the present disclosure are related to use of iPSCs forgeneration of pancreatic α, β and/or δ cells or precursors thereof. By“induced pluripotent stem cell” or “iPSC”, it can be meant a PSC that isderived from a cell that is not a PSC (e.g., from a cell this isdifferentiated relative to a PSC). iPSCs can be derived from multipledifferent cell types, including terminally differentiated cells. iPSCscan have an ES cell-like morphology, growing as flat colonies with largenucleo-cytoplasmic ratios, defined borders and prominent nuclei. Inaddition, iPSCs can express one or more key pluripotency markers knownby one of ordinary skill in the art, including but not limited toAlkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181,TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. Examples ofmethods of generating and characterizing iPSCs can be found in, forexample, U.S. Patent Publication Nos. US20090047263, US20090068742,US20090191159, US20090227032, US20090246875, and US20090304646, each ofwhich are incorporated herein by its entirety. Generally, to generateiPSCs, somatic cells are provided with reprogramming factors (e.g. Oct4,SOX2, KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram thesomatic cells to become pluripotent stem cells.

Embodiments of the present disclosure are related to use of somaticcells for generation of pancreatic α, β and/or δ cells or precursorsthereof. By “somatic cell”, it can be meant any cell in an organismthat, in the absence of experimental manipulation, does not ordinarilygive rise to all types of cells in an organism. In other words, somaticcells can be cells that have differentiated sufficiently that they maynot naturally generate cells of all three germ layers of the body, e.g.ectoderm, mesoderm and endoderm. For example, somatic cells can includeboth neurons and neural progenitors, the latter of which is able tonaturally give rise to all or some cell types of the central nervoussystem but cannot give rise to cells of the mesoderm or endodermlineages

In certain examples, the stem cells can be undifferentiated (e.g. a cellnot committed to a specific lineage) prior to exposure to at least onedifferentiation factor or composition according to the methods asdisclosed herein, whereas in other examples it can be desirable todifferentiate the stem cells to one or more intermediate cell typesprior to exposure of the at least one differentiation factor orcomposition described herein. For example, the stems cells can displaymorphological, biological or physical characteristics ofundifferentiated cells that can be used to distinguish them fromdifferentiated cells of embryo or adult origin. In some examples,undifferentiated cells can appear in the two dimensions of a microscopicview in colonies of cells with high nuclear/cytoplasmic ratios andprominent nucleoli. The stem cells can be themselves (for example,without substantially any undifferentiated cells being present) or canbe used in the presence of differentiated cells. In certain examples,the stem cells can be cultured in the presence of) suitable nutrientsand optionally other cells such that the stem cells can grow andoptionally differentiate. For example, embryonic fibroblasts orfibroblast-like cells can be present in the culture to assist in thegrowth of the stem cells. The fibroblast can be present during one stageof stem cell growth but not necessarily at all stages. For example, thefibroblast can be added to stem cell cultures in a first culturing stageand not added to the stem cell cultures in one or more subsequentculturing stages.

Stem cells used in all aspects of the present invention can be any cellsderived from any kind of tissue (for example embryonic tissue such asfetal or pre-fetal tissue, or adult tissue), which stem cells can havethe characteristic of being capable under appropriate conditions ofproducing progeny of different cell types, e.g. derivatives of all of atleast one of the 3 germinal layers (endoderm, mesoderm, and ectoderm).These cell types can be provided in the form of an established cellline, or they can be obtained directly from primary embryonic tissue andused immediately for differentiation. Included are cells listed in theNIH Human Embryonic Stem Cell Registry, e.g. hESBGN-01, hESBGN-02,hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4,HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-SeoulNational University); HSF-1, FISF-6 (University of California at SanFrancisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni ResearchFoundation (WiCell Research Institute)). In some embodiments, the sourceof human stem cells or pluripotent stem cells used forchemically-induced differentiation into mature, insulin positive cellsdid not involve destroying a human embryo. In some embodiments, thesource of human stem cells or pluripotent stem cells used forchemically-induced differentiation into mature, insulin positive cellsdo not involve destroying a human embryo.

In another example, the stem cells can be isolated from tissue includingsolid tissue. In some embodiments, the tissue is skin, fat tissue (e.g.adipose tissue), muscle tissue, heart or cardiac tissue. In otherembodiments, the tissue is for example but not limited to, umbilicalcord blood, placenta, bone marrow, or chondral.

Stem cells that can be used in the methods provided herein can alsoinclude embryonic cells of various types, exemplified by human embryonicstem (hES) cells, as described by Thomson et al, (1998) Science282:1145; embryonic stem cells from other primates, such as Rhesus stemcells (Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92:7844);marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 55:254); andhuman embryonic germ (hEG) cells (Shambloft et al., Proc. Natl. Acad.Sci. USA 95:13726, 1998). Also applicable to the methods provided hereincan be lineage committed stem cells, such as mesodermal stem cells andother early cardiogenic cells (see Reyes et al, (2001) Blood98:2615-2625; Eisenberg & Bader (1996) Circ Res. 78(2):205-16; etc.) Thestem cells can be obtained from any mammalian species, e.g. human,equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats,hamster, primate, etc. In some embodiments, a human embryo was notdestroyed for the source of pluripotent cell used on the methods andcompositions as disclosed herein. In some embodiments, a human embryo isnot destroyed for the source of pluripotent cell used on the methods andcompositions as disclosed herein.

A mixture of cells from a suitable source of endothelial, muscle, and/orneural stem cells can be harvested from a mammalian donor for thepurpose of the present disclosure. A suitable source is thehematopoietic microenvironment. For example, circulating peripheralblood, preferably mobilized (e.g., recruited), may be removed from asubject. In an embodiment, the stem cells can be reprogrammed stemcells, such as stem cells derived from somatic or differentiated cells.In such an embodiment, the de-differentiated stem cells can be forexample, but not limited to, neoplastic cells, tumor cells and cancercells or alternatively induced reprogrammed cells such as inducedpluripotent stem cells or iPS cells.

In some embodiments, the pancreatic α, β and/or δ cell as describedherein can be derived from one or more of trichocytes, keratinocytes,gonadotropes, corticotropes, thyrotropes, somatotropes, lactotrophs,chromaffin cells, parafollicular cells, glomus cells melanocytes, nevuscells, Merkel cells, odontoblasts, cementoblasts corneal keratocytes,retina Muller cells, retinal pigment epithelium cells, neurons, glias(e.g., oligodendrocyte astrocytes), ependymocytes, pinealocytes,pneumocytes (e.g., type I pneumocytes, and type II pneumocytes), claracells, goblet cells, G cells, D cells, ECL cells, gastric chief cells,parietal cells, foveolar cells, K cells, D cells, I cells, goblet cells,paneth cells, enterocytes, microfold cells, hepatocytes, hepaticstellate cells (e.g., Kupffer cells from mesoderm), cholecystocytes,centroacinar cells, pancreatic stellate cells, pancreatic α cells,pancreatic β cells, pancreatic δ cells, pancreatic F cells (e.g., PPcells), pancreatic ε cells, thyroid (e.g., follicular cells),parathyroid (e.g., parathyroid chief cells), oxyphil cells, urothelialcells, osteoblasts, osteocytes, chondroblasts, chondrocytes,fibroblasts, fibrocytes, myoblasts, myocytes, myosatellite cells, tendoncells, cardiac muscle cells, lipoblasts, adipocytes, interstitial cellsof cajal, angioblasts, endothelial cells, mesangial cells (e.g.,intraglomerular mesangial cells and extraglomerular mesangial cells),juxtaglomerular cells, macula densa cells, stromal cells, interstitialcells, telocytes simple epithelial cells, podocytes, kidney proximaltubule brush border cells, sertoli cells, leydig cells, granulosa cells,peg cells, germ cells, spermatozoon ovums, lymphocytes, myeloid cells,endothelial progenitor cells, endothelial stem cells, angioblasts,mesoangioblasts, pericyte mural cells, splenocytes (e.g., T lymphocytes,B lymphocytes, dendritic cells, microphages, leukocytes), trophoblaststem cells, or any combination thereof.

Reprogramming

The term “reprogramming” as used herein can refer to the process thatalters or reverses the differentiation state of a somatic cell. The cellcan either be partially or terminally differentiated prior to thereprogramming. Reprogramming can encompass complete reversion of thedifferentiation state of a somatic cell to a pluripotent cell. Suchcomplete reversal of differentiation can produce an induced pluripotent(iPS) cell. Reprogramming as used herein can also encompass partialreversion of a cells differentiation state, for example to a multipotentstate or to a somatic cell that is neither pluripotent or multipotent,but is a cell that has lost one or more specific characteristics of thedifferentiated cell from which it arises, e.g. direct reprogramming of adifferentiated cell to a different somatic cell type. Reprogramming caninvolve alteration, e.g., reversal, of at least some of the heritablepatterns of nucleic acid modification (e.g., methylation), chromatincondensation, epigenetic changes, genomic imprinting, etc., that occurduring cellular differentiation as a zygote develops into an adult.

As used herein, the term “reprogramming factor” can refer to a moleculethat is associated with cell “reprogramming,” that is, differentiation,and/or de-differentiation, and/or transdifferentiation, such that a cellconverts to a different cell type or phenotype. Reprogramming factorsgenerally affect expression of genes associated with celldifferentiation, de-differentiation and/or transdifferentiation.Transcription factors are examples of reprogramming factors.

The term “differentiation” and their grammatical equivalents as usedherein can refer to the process by which a less specialized cell (e.g.,a more naive cell with a higher cell potency) becomes a more specializedcell type (e.g., a less naive cell with a lower cell potency); and thatthe term “de-differentiation” can refer to the process by which a morespecialized cell becomes a less specialized cell type (e.g., a morenaive cell with a higher cell potency); and that the term“transdifferentiation” refers to the process by which a cell of aparticular cell type converts to another cell type without significantlychanging its “cell potency” or “naivety” level. Without wishing to bebound by theory, it is thought that cells “transdifferentiate” when theyconvert from one lineage-committed cell type or terminallydifferentiated cell type to another lineage-committed cell type orterminally differentiated cell type, without significantly changingtheir “cell potency” or “naivety” level.

As used herein, the term “cell potency” is to be understood as referringto the ability of a cell to differentiate into cells of differentlineages. For example, a pluripotent cell (e.g., a stem cell) has thepotential to differentiate into cells of any of the three germ layers,that is, endoderm (interior stomach lining, gastrointestinal tract, thelungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm(epidermal tissues and nervous system), and accordingly has high cellpotency; a multipotent cell (e.g., a stem cell or an induced stem cellof a certain type) has the ability to give rise to cells from amultiple, but limited, number of lineages (such as hematopoietic stemcells, cardiac stem cells, or neural stem cells, etc.) comparatively hasa lower cell potency than pluripotent cells. Cells that are committed toa particular lineage or are terminally differentiated can have yet alower cell potency. Specific examples of transdifferentiation known inthe art include the conversion of e.g., fibroblasts beta cells or frompancreatic exocrine cells to beta cells etc.

Accordingly, the cell may be caused to differentiate into a more naivecell (e.g., a terminally differentiated cell may be differentiated to bemultipotent or pluripotent); or the cell may be caused tode-differentiate into a less naive cell (e.g., a multipotent orpluripotent cell can be differentiated into a lineage-committed cell ora terminally differentiated cell). However, in an embodiment, the cellmay be caused to convert or transdifferentiate from one cell type (orphenotype) to another cell type (or phenotype), for example, with asimilar cell potency level. Accordingly, in an embodiment of the presentdisclosure, the inducing steps of the present disclosure can reprogramthe cells of the present disclosure to differentiate, de-differentiateand/or transdifferentiate. In an embodiment of the present disclosure,the inducing steps of the present disclosure may reprogram the cells totransdifferentiate.

Methods of reprogramming or inducing a particular type of cell to becomeanother type of cell, for example, by differentiation,de-differentiation and/or transdifferentiation using one or moreexogenous polynucleotide or polypeptide reprogramming factors are knownto the person skilled in the art. Such methods may rely on theintroduction of genetic material encoding one or more transcriptionfactor(s) or other polypeptide(s) associated with cell reprogramming.For example, PDX1, Ngn3 and MafA, or functional fragments thereof areall known to encode peptides that can induce cell differentiation,de-differentiation and/or transdifferentiation of the cells of thepresent disclosure. In some methods known to the person skilled in theart, exogenous polypeptides (e.g. recombinant polypeptides) encoded byreprogramming genes (such as the above genes) are contacted with thecells to induce, for example, cells of the present disclosure. Theperson skilled in the art will appreciate that other genes may beassociated with reprogramming of cells, and exogenous molecules encodingsuch genes (or functional fragments thereof) and the encodedpolypeptides are also considered to be polynucleotide or polypeptidereprogramming factors (e.g. polynucleotides or polypeptides that in turnaffect expression levels of another gene associated with cellreprogramming). For example, it has been shown that the introduction ofexogenous polynucleotide or polypeptide epigenetic gene silencers thatdecrease p53 inactivation increase the efficiency of inducing inducedpluripotent stem cells (iPSC). Accordingly, exogenous polynucleotides orpolypeptides encoding epigenetic silencers and other genes or proteinsthat may be directly or indirectly involved in cell reprogramming orincreasing cell programming efficiency would be considered to constitutean exogenous polynucleotide or polypeptide reprogramming factor. Theperson skilled in the art will appreciate that other methods ofinfluencing cell reprogramming exist, such as introducing RNAi molecules(or genetic material encoding RNAi molecules) that can knock downexpression of genes involved in inhibiting cell reprogramming.Accordingly, any exogenous polynucleotide molecule or polypeptidemolecule that is associated with cell reprogramming, or enhances cellreprogramming, is to be understood to be an exogenous polynucleotide orpolypeptide reprogramming factor as described herein.

In some embodiments of the present disclosure, the method excludes theuse of reprogramming factor(s) that are not small molecules. However, itwill be appreciated that the method can utilize “routine” tissue culturecomponents such as culture media, serum, serum substitutes, supplements,antibiotics, etc, such as RPMI, Renal Epithelial Basal Medium (REBM),Dulbecco's Modified Eagle Medium (DMEM), MCDB131 medium, CMRL 1066medium, F12, foetal calf serum (FCS), foetal bovine serum (FBS), bovineserum albumin (BSA), D-glucose, L-glutamine, GlutaMAX.TM.-1 (dipeptide,L-alanine-L-glutamine), B27, heparin, progesterone, putrescine, laminin,nicotinamide, insulin, transferrin, sodium selenite, selenium,ethanolamine, human epidermal growth factor (hEGF), basic fibroblastgrowth factor (bFGF), hydrocortisone, epinephrine, normacin, penicillin,streptomycin, gentamicin and amphotericin, etc. It is to be understoodthat these typical tissue culture components (and other similar tissueculture components that are routinely used in tissue culture) are notsmall molecule reprogramming molecules for the purposes of the presentdisclosure. These components are either not small molecules as definedherein and/or are not reprogramming factors as defined herein.

Accordingly, in an embodiment, the present disclosure does not involve aculturing step of the cell(s) with one or more exogenous polynucleotideor polypeptide reprogramming factor(s). Accordingly, in an embodiment,the method of the present disclosure does not involve the introductionof one or more exogenous polynucleotide or polypeptide reprogrammingfactor(s), e.g., by introducing transposons, viral transgenic vectors(such as retroviral vectors), plasmids, mRNA, miRNA, peptides, orfragments of any of these molecules, that are involved in producinginduced α, β and/or δ cells or, otherwise, inducing cells of the presentdisclosure to differentiate, de-differentiation and/ortransdifferentiate.

That is, in an embodiment, the method occurs in the absence of one ormore exogenous polynucleotide or polypeptide reprogramming factor(s).Accordingly, it is to be understood that in an embodiment, the method ofthe present disclosure utilizes small molecules (e.g., HDAC inhibitors)to reprogram cells, without the addition of polypeptide transcriptionfactors; other polypeptide factors specifically associated with inducingdifferentiation, de-differentiation, and/or transdifferentiation;polynucleotide sequences encoding polypeptide transcription factors,polynucleotide sequences encoding other polypeptide factors specificallyassociated with inducing differentiation, de-differentiation, and/ortransdifferentiation; mRNA; interference RNA; microRNA and fragmentsthereof.

Methods of Generating Stem Cell Derived β Cells

Provided herein are methods of generating SC-β cells (e.g., non-nativepancreatic β cells). The detailed protocols of generating endocrinecells the stem cells to provide at least one SC-β cell are described inU.S. Patent Application Publication No. US20150240212 and US20150218522,each of which is herein incorporated by reference in its entirety.

The endoderm can give rise to digestive and respiratory tracts, thyroid,liver, and pancreas. Representative disease of endoderm lineages is type1 diabetes resulting from destruction of the insulin-producing β cells.Generation of functional β cells from human pluripotent stem cells(hPSC) in vitro can be practical, renewable cell source for replacementcell therapy for type 1 diabetes. The embryotic stem (ES) cells that aregenerated from the inner cell mass of blastocyst-stage embryos representa promising source of cells for transplantation or cell-based therapy ofany damaged cells. They can be maintained in culture, renew forthemselves, and proliferate unlimitedly as undifferentiated ES cells.The ES cells are capable of differentiating into all cell types of thebody as the ectoderm, mesoderm, and endoderm lineage cells or tissues.The major benefit of ES cells is stable self-renewal in culture and thepotential to differentiate.

The definitive endoderm can be generated in vivo from the inner cellmass by the process of gastrulation of embryogenesis, in which epiblastcells are instructed to form the three germ layers. Definitive endodermcan give rise to diverse cells and tissues that contribute to vitalorgans as the pancreatic β cells, liver hepatocytes, lung alveolarcells, thyroid, thymus, and the epithelial lining of the alimentary andrespiratory tract. It is different from the primitive endoderm ofextraembryonic tissues, which can give rise to the visceral and parietalendoderm. The definitive endoderm derived from ES cells is theoreticallycapable of becoming any endoderm derivatives, and directing ES cellsinto the endoderm lineage is a prerequisite for generating therapeuticendoderm derivatives.

Precise patterning of anterior-posterior axis of the definitive endodermcan eventually form the primitive gut tube. The definitiveendoderm-derived primitive gut tube induces the pharynx, esophagus,stomach, duodenum, small and large intestine along theanterior-posterior axis as well as associated organs, includingpancreas, lung, thyroid, thymus, parathyroid, and liver. The anteriorportion of the foregut of the primitive gut tube becomes lung, thyroid,esophagus, and stomach. The pancreas, liver, and duodenum originate fromthe posterior portion of the foregut. The midgut and hindgut ofprimitive gut tube gives rise to the small and large intestine. Theanterior foregut expresses developmental markers, NK2 homeobox 1(NKX2-1) and SRY (sex determining region Y)-box 2 (50X2); the posteriorforegut expresses hematopoietically expressed homeobox (HHEX),pancreatic and duodenal homeobox 1 (PDX1), one cut homeobox 1 (ONECUT1,known as HNF6), and hepatocyte nuclear factor 4 alpha (HNF4A); and themidgut/hindgut expresses caudal type homeobox 1 (CDX1), caudal typehomeobox 2 (CDX2), and motor neuron and pancreas homeobox 1 (MNX1) (3,19, 20).

The successful differentiation to pancreatic β cells should require thatdifferentiated cells synthesize and secrete physiologically appropriateamounts of insulin. An exemplary stepwise protocol directing hPSC celldifferentiation is developed, which entails differentiation processesthat recapitulates the major stages of normal pancreatic endocrinedevelopment (for instance, the Version A protocol in EXAMPLE 1). Thedifferentiation of hPSC cells to hormone-expressing pancreatic endocrinecells is conducted by transiting hPSC cells through major stages ofembryonic development; differentiation to mesendoderm and definitiveendoderm, establishment of the primitive gut endoderm, patterning of theposterior foregut, and specification and maturation of pancreaticendoderm and endocrine precursors. Through these stages, hPSC cells canobtain pancreatic endocrine phenotype and ability of glucose responsiveinsulin secretion in vitro.

Generally, the at least one pancreatic α, β and/or δ cell or precursorthereof, e.g., pancreatic progenitors produced according to the methodsdisclosed herein can comprise a mixture or combination of differentcells, e.g., for example a mixture of cells such as a PDX1-positivepancreatic progenitors, pancreatic progenitors co-expressing PDX1 andNKX6-1, a Ngn3-positive endocrine progenitor cell, an insulin-positiveendocrine cell (e.g., NKX6.1-positive, ISL1-positive cells, or β-likecells), and/or other pluripotent or stem cells.

The at least one pancreatic α, β and/or δ cell or precursor thereof canbe produced according to any suitable culturing protocol todifferentiate a stem cell or pluripotent cell to a desired stage ofdifferentiation. In some embodiments, the at least one pancreatic α, βand/or δ cell or the precursor thereof are produced by culturing atleast one pluripotent cell for a period of time and under conditionssuitable for the at least one pluripotent cell to differentiate into theat least one pancreatic α, β and/or δ cell or the precursor thereof.

In some embodiments, the at least one pancreatic α, β and/or δ cell orprecursor thereof is a substantially pure population of pancreatic α, βand/or δ cells or precursors thereof. In some embodiments, a populationof pancreatic α, β and/or δ cells or precursors thereof comprises amixture of pluripotent cells or differentiated cells. In someembodiments, a population pancreatic α, β and/or δ cells or precursorsthereof are substantially free or devoid of embryonic stem cells orpluripotent cells or iPS cells.

In some embodiments, a somatic cell, e.g., fibroblast can be isolatedfrom a subject, for example as a tissue biopsy, such as, for example, askin biopsy, and reprogrammed into an induced pluripotent stem cell forfurther differentiation to produce the at least one pancreatic α, βand/or δ cell or precursor thereof for use in the compositions andmethods described herein. In some embodiments, a somatic cell, e.g.,fibroblast is maintained in culture by methods known by one of ordinaryskill in the art, and in some embodiments, propagated prior to beingconverted into pancreatic α, β and/or δ cells by the methods asdisclosed herein.

In some embodiments, the at least one pancreatic α, β and/or δ cell orprecursor thereof are maintained in culture by methods known by one ofordinary skill in the art, and in some embodiments, propagated prior tobeing converted into pancreatic α, β and/or δ cells by the methods asdisclosed herein.

Further, at least one pancreatic α, β and/or δ cell or precursorthereof, e.g., pancreatic progenitor can be from any mammalian species,with non-limiting examples including a murine, bovine, simian, porcine,equine, ovine, or human cell. For clarity and simplicity, thedescription of the methods herein refers to a mammalian at least onepancreatic α, β and/or δ cell or precursor thereof but it should beunderstood that all of the methods described herein can be readilyapplied to other cell types of at least one pancreatic α, β and/or δcell or precursor thereof. In some embodiments, the at least one SC-βcell or precursor thereof is derived from a human individual.

Definitive Endoderm Cells

Aspects of the disclosure involve definitive endoderm cells. Definitiveendoderm cells of use herein can be derived from any source or generatedin accordance with any suitable protocol. In some aspects, pluripotentstem cells, e.g., iPSCs or hESCs, are differentiated to endoderm cells.In some aspects, the endoderm cells (stage 1) are furtherdifferentiated, e.g., to primitive gut tube cells (stage 2),PDX1-positive pancreatic progenitor cells (stage 3), NKX6.1-positivepancreatic progenitor cells (stage 4), or Ngn3-positive endocrineprogenitor cells or insulin-positive endocrine cells (stage 5), followedby induction or maturation to SC-β cells (stage 6).

In some cases, definitive endoderm cells can be obtained bydifferentiating at least some pluripotent cells in a population intodefinitive endoderm cells, e.g., by contacting a population ofpluripotent cells with i) at least one growth factor from the TGF-βsuperfamily, and ii) a WNT signaling pathway activator, to induce thedifferentiation of at least some of the pluripotent cells intodefinitive endoderm cells, wherein the definitive endoderm cells expressat least one marker characteristic of definitive endoderm.

Any growth factor from the TGF-β superfamily capable of inducing thepluripotent stem cells to differentiate into definitive endoderm cells(e.g., alone, or in combination with a WNT signaling pathway activator)can be used in the method provided herein. In some cases, the growthfactor from the TGF-β superfamily comprises Activin A. In some cases,the growth factor from the TGF-β superfamily comprises growthdifferentiating factor 8 (GDF8). Any WNT signaling pathway activatorcapable of inducing the pluripotent stem cells to differentiate intodefinitive endoderm cells (e.g., alone, or in combination with a growthfactor from the TGF-β superfamily) can be used in the method providedherein. In some cases, the WNT signaling pathway activator comprisesCHIR99Q21. In some cases, the WNT signaling pathway activator comprisesWnt3a recombinant protein.

In some cases, differentiating at least some pluripotent cells in apopulation into definitive endoderm cells is achieved by a process ofcontacting a population of pluripotent cells with i) Activin A, and ii)CHIR99021 for a suitable period of time, e.g., about 2 days, about 3days, about 4 days, or about 5 days to induce the differentiation of atleast some of the pluripotent cells in the population into definitiveendoderm cells, wherein the definitive endoderm cells express at leastone marker characteristic of definitive endoderm.

In some examples, the method comprises differentiating pluripotent cellsinto definitive endoderm cells by contacting a population of pluripotentcells with a suitable concentration of the growth factor from the TGF-βsuperfamily (e.g., Activin A), such as, about 10 ng/mL, about 20 ng/mL,about 50 ng/mL, about 75 ng/mL, about 80 ng/mL, about 90 ng/mL, about 95ng/mL, about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130ng/mL, about 140 ng/mL, about 150 ng/mL, about 175 ng/mL, about 180ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL. In somecases, the method comprises use of about 100 ng/mL Activin A fordifferentiation of pluripotent cells into definitive endoderm cells. Insome cases, the method comprises use of about 200 ng/mL Activin A fordifferentiation of pluripotent cells into definitive endoderm cells.

In some examples, the method comprises differentiating pluripotent cellsinto definitive endoderm cells by contacting a population of pluripotentcells with a suitable concentration of the WNT signaling pathwayactivator (e.g., CHIR99021), such as, about 0.01 μM, about 0.05 μM,about 0.1 μM, about 0.2 μM, about 0.5 μM, about 0.8 μM, about 1 μM,about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about4 μM, about 5 μM, about 8 μM, about 10 μM, about 12 μM, about 15 μM,about 20 μM, about 30 μM, about 50 μM, about 100 μM, or about 200 μM. Insome cases, the method comprises use of about 2 μM CHIR99021 fordifferentiation of pluripotent cells into definitive endoderm cells. Insome cases, the method comprises use of about 5 μM CHIR99021 fordifferentiation of pluripotent cells into definitive endoderm cells.

In some cases, a definitive endoderm cell produced by the methods asdisclosed herein expresses at least one marker selected from the groupconsisting of: Noda1, Tmprss2, Tmem30b, St14, Spink3, Sh3g12, Ripk4,Rab1S, Npnt, Clic6, Cldn5, Cacnalb, Bnipl, Anxa4, Emb, FoxA1, Sox17, andRbm35a, wherein the expression of at least one marker is upregulated toby a statistically significant amount in the definitive endoderm cellrelative to the pluripotent stem cell from which it was derived. In somecases, a definitive endoderm cell produced by the methods as disclosedherein does not express by a statistically significant amount at leastone marker selected the group consisting of: Gata4, SPARC, AFP and Dab2relative to the pluripotent stem cell from which it was derived. In somecases, a definitive endoderm cell produced by the methods as disclosedherein does not express by a statistically significant amount at leastone marker selected the group consisting of: Zic1, Pax6, Flk1 and CD31relative to the pluripotent stem cell from which it was derived. In somecases, a definitive endoderm cell produced by the methods as disclosedherein has a higher level of phosphorylation of Smad2 by a statisticallysignificant amount relative to the pluripotent stem cell from which itwas derived. In some cases, a definitive endoderm cell produced by themethods as disclosed herein has the capacity to form gut tube in vivo.In some cases, a definitive endoderm cell produced by the methods asdisclosed herein can differentiate into a cell with morphologycharacteristic of a gut cell, and wherein a cell with morphologycharacteristic of a gut cell expresses FoxA2 and/or Claudin6, In somecases, a definitive endoderm cell produced by the methods as disclosedherein can be further differentiated into a cell of endoderm origin.

In some cases, a population of pluripotent stem cells are cultured inthe presence of at least one β cell differentiation factor prior to anydifferentiation or during the first stage of differentiation. One canuse any pluripotent stem cell, such as a human pluripotent stem cell, ora human iPS cell or any of pluripotent stem cell as discussed herein orother suitable pluripotent stem cells. In some cases, a β celldifferentiation factor as described herein can be present in the culturemedium of a population of pluripotent stem cells or may be added inbolus or periodically during growth (e.g. replication or propagation) ofthe population of pluripotent stem cells. In certain examples, apopulation of pluripotent stem cells can be exposed to at least one βcell differentiation factor prior to any differentiation. In otherexamples, a population of pluripotent stem cells may be exposed to atleast one β cell differentiation factor during the first stage ofdifferentiation.

Primitive Gut Tube Cells

Aspects of the disclosure involve primitive gut tube cells. Primitivegut tube cells of use herein can be derived from any source or generatedin accordance with any suitable protocol. In some aspects, definitiveendoderm cells are differentiated to primitive gut tube cells. In someaspects, the primitive gut tube cells are further differentiated, e.g.,to PDX1-positive pancreatic progenitor cells, NKX6.1-positive pancreaticprogenitor cells, Ngn3-positive endocrine progenitor cells,insulin-positive endocrine cells, followed by induction or maturation toSC-β cells.

In some cases, primitive gut tube cells can be obtained bydifferentiating at least some definitive endoderm cells in a populationinto primitive gut tube cells, e.g., by contacting definitive endodermcells with at least one growth factor from the fibroblast growth factor(FGF) family, to induce the differentiation of at least some of thedefinitive endoderm cells into primitive gut tube cells, wherein theprimitive gut tube cells express at least one marker characteristic ofprimitive gut tube cells.

Any growth factor from the FGF family capable of inducing definitiveendoderm cells to differentiate into primitive gut tube cells (e.g.,alone, or in combination with other factors) can be used in the methodprovided herein. In some cases, the at least one growth factor from theFGF family comprises keratinocyte growth factor (KGF). In some cases,the at least one growth factor from the FGF family comprises FGF2. Insome cases, the at least one growth factor from the FGF family comprisesFGF8B. In some cases, the at least one growth factor from the FGF familycomprises FGF 10. In some cases, the at least one growth factor from theFGF family comprises FGF21.

In some cases, primitive gut tube cells can be obtained bydifferentiating at least some definitive endoderm cells in a populationinto primitive gut tube cells, e.g., by contacting definitive endodermcells with KGF for a certain period of time, e.g., about 1 day, about 2days, about 3 days, or about 4 days, to induce the differentiation of atleast some of the definitive endoderm cells into primitive gut tubecells.

In some cases, the method comprises differentiating definitive endodermcells into primitive gut tube cells by contacting definitive endodermcells with a suitable concentration of the growth factor from the FGFfamily (e.g., KGF), such as, about 10 ng/mL, about 20 ng/mL, about 50ng/mL, about 75 ng/mL, about 80 ng/mL, about 90 ng/mL, about 95 ng/mL,about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130 ng/mL,about 140 ng/mL, about 150 ng/mL, about 175 ng/mL, about 180 ng/mL,about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL. In some cases, themethod comprises use of about 50 ng/mL KGF for differentiation ofdefinitive endoderm cells into primitive gut tube cells. In some cases,the method comprises use of about 100 ng/mL KGF for differentiation ofdefinitive endoderm cells into primitive gut tube cells.

DX1-Positive Pancreatic Progenitor Cells

Aspects of the disclosure involve PDX1-positive pancreatic progenitorcells. PDX1-positive pancreatic progenitor cells of use herein can bederived from any source or generated in accordance with any suitableprotocol. In some aspects, primitive gut tube cells are differentiatedto PDX1-positive pancreatic progenitor cells. In some aspects, thePDX1-positive pancreatic progenitor cells are further differentiated,e.g., NKX6.1-positive pancreatic progenitor cells, Ngn3-positiveendocrine progenitor cells, insulin-positive endocrine cells, followedby induction or maturation to SC-β cells,

In some aspects, PDX1-positive pancreatic progenitor cells can beobtained by differentiating at least some primitive gut tube cells in apopulation into PDX1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with i) at least one BMP signalingpathway inhibitor, ii) a growth factor from TGF-β superfamily, iii) atleast one growth factor from the FGF family, iv) at least one SHHpathway inhibitor, v) at least one retinoic acid (RA) signaling pathwayactivator; vi) at least one protein kinase C activator, and vii) ROCKinhibitor to induce the differentiation of at least some of theprimitive gut tube cells into PDX1-positive pancreatic progenitor cells,wherein the PDX1-positive pancreatic progenitor cells express PDX1.

In some aspects, PDX1-positive pancreatic progenitor cells can beobtained by differentiating at least some primitive gut tube cells in apopulation into PDX1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with i) at least one BMP signalingpathway inhibitor, ii) a growth factor from TGF-β superfamily, iii) atleast one growth factor from the FGF family, iv) at least one SHHpathway inhibitor, v) at least one retinoic acid (RA) signaling pathwayactivator; and vi) at least one protein kinase C activator, to inducethe differentiation of at least some of the primitive gut tube cellsinto PDX1-positive pancreatic progenitor cells, wherein thePDX1-positive pancreatic progenitor cells express PDX1.

In some cases, PDX1-positive pancreatic progenitor cells can be obtainedby differentiating at least some primitive gut tube cells in apopulation into PDX1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with i) at least one BMP signalingpathway inhibitor, ii) at least one growth factor from the FGF family,iii) at least one SHH pathway inhibitor, iv) at least one retinoic acid(RA) signaling pathway activator; and v) at least one protein kinase Cactivator, to induce the differentiation of at least some of theprimitive gut tube cells into PDX1-positive pancreatic progenitor cells,wherein the PDX1-positive pancreatic progenitor cells express PDX1.

In some cases, PDX1-positive pancreatic progenitor cells can be obtainedby differentiating at least some primitive gut tube cells in apopulation into PDX1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with i) at least one SHH pathwayinhibitor, ii) at least one retinoic acid (RA) signaling pathwayactivator; and iii) at least one protein kinase C activator, wherein thePDX1-positive pancreatic progenitor cells express PDX1.

In some cases, PDX1-positive pancreatic progenitor cells can be obtainedby differentiating at least some primitive gut tube cells in apopulation into PDX1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with i) at least one growth factorfrom the FGF family, and ii) at least one retinoic acid (RA) signalingpathway activator, to induce the differentiation of at least some of theprimitive gut tube cells into PDX1-positive pancreatic progenitor cells,wherein the PDX1-positive pancreatic progenitor cells express PDX1.

Any BMP signaling pathway inhibitor capable of inducing primitive guttube cells to differentiate into PDX1-positive pancreatic progenitorcells (e.g., alone, or with any combination of a growth factor fromTGF-β superfamily, at least one growth factor from the FGF family, atleast one SHH pathway inhibitor, at least one retinoic acid signalingpathway activator, at least one protein kinase C activator, and ROCKinhibitor) can be used in the method provided herein. In some cases, theBMP signaling pathway inhibitor comprises LDN193189 or DMH-1. In someexamples, the method comprises contacting primitive gut tube cells witha concentration of BMP signaling pathway inhibitor (e.g., LDN1931189),such as, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM,about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM,about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM,about 230 nM, about 240 nM, about 250 nM, about 280 nM, about 300 nM,about 400 nM, about 500 nM, or about 1 μM. In some examples, the methodcomprises contacting primitive gut tube cells with a concentration ofBMP signaling pathway inhibitor (e.g., DMH-1), such as, about 0.01 μM,about 0.02 μM about 0.05 μM about 0.1 μM, about 0.2 μM about 0.5 μM,about 0.8 μM, about 1 μM, about 1.2 μM, about 1.5 μM about 1.75 μM about2 μM, about 2.2 μM, about 2.5 μM about 2.75 μM, about 3 μM, about 3.25μM, about 3.5 μM, about 3.75 μM, about 4 μM, about 4.5 μM, about 5 μM,about 8 μM, about 10 μM, about 15 μM, about 20 μM, about 30 μM, about 40μM, about 50 μM, or about 100 μM.

Any growth factor from the TGF-β superfamily capable of inducingprimitive gut tube cells to differentiate into PDX1-positive pancreaticprogenitor cells (e.g., alone, or with any combination of at least oneBMP signaling pathway inhibitor, a growth factor from the FGF family, atleast one SHH pathway inhibitor, at least one retinoic acid signalingpathway activator, at least one protein kinase C activator, and ROCKinhibitor) can be used. In some cases, the growth factor from TGF-βfamily comprises Activin A. In some cases, the growth factor from TGF-βfamily comprises Activin A or GDF8. In some examples, the methodcomprises contacting primitive gut tube cells with a concentration of agrowth factor from TGF-β superfamily (e.g., Activin A), such as, about 5ng/mL, about 7.5 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL,about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL,about 20 ng/mL, about 21 ng/mL, about 22 ng/mL, about 23 ng/mL, about 24ng/mL, about 25 ng/mL, about 26 ng/mL, about 27 ng/mL, about 28 ng/mL,about 29 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50ng/mL, or about 100 ng/mL.

Any growth factor from the FGF family capable of inducing primitive guttube cells to differentiate into PDX1-positive pancreatic progenitorcells (e.g., alone, or with any combination of at least one BMPsignaling pathway inhibitor, a growth factor from TGF-β superfamily, atleast one SHH pathway inhibitor, at least one retinoic acid signalingpathway activator, at least one protein kinase C activator, and ROCKinhibitor) can be used. In some cases, the at least one growth factorfrom the FGF family comprises keratinocyte growth factor (KGF). In somecases, the at least one growth factor from the FGF family is selectedfrom the group consisting of FGF2, FGF8B, FGF 10, and FGF21. In someexamples, the method comprises contacting primitive gut tube cells witha concentration of a growth factor from FGF family (e.g., KGF), such as,about 10 ng/mL, about 20 ng/mL, about 50 ng/mL, about 75 ng/mL, about 80ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 110 ng/mL,about 120 ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL,about 175 ng/mL, about 180 ng/mL, about 200 ng/mL, about 250 ng/mL, orabout 300 ng/mL.

Any SHH pathway inhibitor capable of inducing primitive gut tube cellsto differentiate into PDX1-positive pancreatic progenitor cells (e.g.,alone, or with any combination of at least one BMP signaling pathwayinhibitor, at least one growth factor from the FGF family, a growthfactor from TGF-β superfamily, at least one retinoic acid signalingpathway activator, at least one protein kinase C activator, and ROCKinhibitor) can be used. In some cases, the SHH pathway inhibitorcomprises Sant1. In some examples, the method comprises contactingprimitive gut tube cells with a concentration of a SHH pathway inhibitor(e.g., Sant1), such as, about 0.001 μM, about 0.002 μM, about 0.005 μM,about 0.01 μM, about 0.02 μM, about 0.03 μM, about 0.05 μM about 0.08μM, about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM,about 0.2 μM, about 0.21 μM, about 0.22 μM, about 0.23 μM, about 0.24μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about0.29 μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM,about 0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM,about 0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM.

Any RA signaling pathway activator capable of inducing primitive guttube cells to differentiate into PDX1-positive pancreatic progenitorcells (e.g., alone, or with any combination of at least one BMPsignaling pathway inhibitor, at least one growth factor from the FGFfamily, at least one SHH pathway inhibitor, at least one protein kinaseC activator, and ROCK inhibitor) can be used. In some cases, the RAsignaling pathway activator comprises retinoic acid. In some examples,the method comprises contacting primitive gut tube cells with aconcentration of an RA signaling pathway activator (e.g., retinoicacid), such as, about 0.02 μM, about 0.1 μM, about 0.2 μM, about 0.25μM, about 0.3 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.55μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM, about 0.8μM, about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM,about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM,about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM,about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM,about 2.8 μM, about 3 μM, about 3.2 μM, about 3.4 μM, about 3.6 μM,about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM, about 4.6 μM,about 4.8 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM,about 10 μM, about 12 μM, about 14 μM, about 15 μM, about 16 μM, about18 μM, about 20 μM, about 50 μM, or about 100 μM.

Any PKC activator capable of inducing primitive gut tube cells todifferentiate into PDX1-positive pancreatic progenitor cells (e.g.,alone, or with any combination of at least one BMP signaling pathwayinhibitor, at least one growth factor from the FGF family, at least oneSHH pathway inhibitor, at least one RA signaling pathway activator, andROCK inhibitor) can be used. In some cases, the PKC activator comprisesPdBU. In some cases, the PKC activator comprises TPPB. In some examples,the method comprises contacting primitive gut tube cells with aconcentration of a PKC activator (e.g., PdBU or TPPB), such as, about 10nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900nM, 950 nM, 1 μM, 10 μM, about 20 μM, about 50 μM, about 75 μM, about 80μM, about 100 μM, about 120 μM, about 140 μM, about 150 μM, about 175μM, about 180 μM, about 200 μM, about 210 μM, about 220 μM, about 240μM, about 250 μM, about 260 μM, about 280 μM, about 300 μM, about 320μM, about 340 μM, about 360 μM, about 380 μM, about 400 μM, about 420μM, about 440 μM, about 460 μM, about 480 μM, about 500 μM, about 520μM, about 540 μM, about 560 μM, about 580 μM, about 600 μM, about 620μM, about 640 μM, about 660 μM, about 680 μM, about 700 μM, about 750μM, about 800 μM, about 850 μM, about 900 μM, about 1 mM, about 2 mM,about 3 mM, about 4 mM, or about 5 mM. In some embodiments, the methodcomprises contacting primitive gut tube cells with a concentration of aPKC activator (e.g., PdBU or TPPB) of 10 nM-1 mM, 10 nM-500 μM, 10 nM-1μM, 10-800 nM, 100-900 nM, 300-800 nM, 300-600 nM, 400-600 nM, 450-550nM, or about 500 nM. In some embodiments, primitive gut tube cells arenot treated with a PKC activator (e.g., PDBU).

Any ROCK inhibitor capable of inducing primitive gut tube cells todifferentiate into PDX1-positive pancreatic progenitor cells (e.g.,alone, or with any combination of at least one BMP signaling pathwayinhibitor, at least one growth factor from the FGF family, at least oneSHH pathway inhibitor, PKC activator, and at least one RA signalingpathway activator) can be used. In some cases, the ROCK inhibitorcomprises Thiazovivin, Y-27632, Fasudil/HA1077, or H-1152. In somecases, the ROCK inhibitor comprises Y-27632. In some cases, the ROCKinhibitor comprises Thiazovivin. In some examples, the method comprisescontacting primitive gut tube cells with a concentration of a ROCKinhibitor (e.g., Y-27632 or Thiazovivin), such as, about 0.2 μM, about0.5 μM, about 0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM,about 5 μM, about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM,about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM,about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about35 μM, about 40 μM, about 50 μM, or about 100 μM.

In some cases, PDX1-positive pancreatic progenitor cells can be obtainedby differentiating at least some primitive gut tube cells in apopulation into PDX1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with retinoic acid, KGF, Sant1,DMH-1, PdBU, thiazovivin, and Activin A, for a suitable period of time,e.g., about 1 day, about 2 days, about 3 days, or about 4 days. In somecases, PDX1-positive pancreatic progenitor cells can be obtained bydifferentiating at least some primitive gut tube cells in a populationinto PDX1-positive pancreatic progenitor cells, e.g., by contactingprimitive gut tube cells with retinoic acid, KGF, Sant1, DMH-1, PdBU,thiazovivin, and Activin A, for about 2 days.

NKX6.1-Positive Pancreatic Progenitor Cells

Aspects of the disclosure involve NKX6.1-positive pancreatic progenitorcells. NKX6.1-positive pancreatic progenitor cells of use herein can bederived from any source or generated in accordance with any suitableprotocol. In some aspects, PDX1-positive pancreatic progenitor cells aredifferentiated to NKX6.1-positive pancreatic progenitor cells. In someaspects, the NKX6.1-positive pancreatic progenitor cells are furtherdifferentiated, e.g., to Ngn3-positive endocrine progenitor cells, orinsulin-positive endocrine cells, followed by induction or maturation toSC-β cells.

In some aspects, a method of producing a NKX6.1-positive pancreaticprogenitor cell from a PDX1-positive pancreatic progenitor cellcomprises contacting a population of cells (e.g., under conditions thatpromote cell clustering and/or promoting cell survival) comprisingPDX1-positive pancreatic progenitor cells with at least two βcell-differentiation factors comprising a) at least one growth factorfrom the fibroblast growth factor (FGF) family, b) a sonic hedgehogpathway inhibitor, and optionally c) a low concentration of a retinoicacid (RA) signaling pathway activator, to induce the differentiation ofat least one PDX1-positive pancreatic progenitor cell in the populationinto NKX6.1-positive pancreatic progenitor cells, wherein theNKX6.1-positive pancreatic progenitor cells expresses NKX6.1.

In some cases, the PDX1-positive, NKX6.1-positive pancreatic progenitorcells are obtained by contacting PDX1-positive pancreatic progenitorcells with i) at least one growth factor from the FGF family, ii) atleast one SHH pathway inhibitor, and optionally iii) a low concentrationof a RA signaling pathway activator, to induce the differentiation of atleast some of the PDX1-positive pancreatic progenitor cells intoPDX1-positive, NKX6.1-positive pancreatic progenitor cells, wherein thePDX1-positive, NKX6.1-positive pancreatic progenitor cells expressesPDX1 and NKX6.1.

In some cases, the PDX1-positive, NKX6.1-positive pancreatic progenitorcells are obtained by contacting PDX1-positive pancreatic progenitorcells with i) at least one growth factor from the FGF family, ii) atleast one SHH pathway inhibitor, and optionally iii) a low concentrationof a RA signaling pathway activator, iv) ROCK inhibitor, and v) at leastone growth factor from the TGF-β superfamily, to induce thedifferentiation of at least some of the PDX1-positive pancreaticprogenitor cells into PDX1-positive, NKX6.1-positive pancreaticprogenitor cells. In some embodiments, following 3, 4, or 5 days ofcontacting the PDX1-positive, NKX6.1-positive pancreatic progenitorcells are obtained by contacting PDX1-positive pancreatic progenitorcells with i) at least one growth factor from the FGF family, ii) atleast one SHH pathway inhibitor, and optionally iii) a low concentrationof a RA signaling pathway activator, iv) ROCK inhibitor, and v) at leastone growth factor from the TGF-β superfamily; the cells are thencontacted with i) at least one growth factor from the FGF family, ii) atleast one SHH pathway inhibitor, and optionally iii) a low concentrationof a RA signaling pathway activator, iv) ROCK inhibitor, and v) at leastone growth factor from the TGF-β superfamily, and vi) a PKC activatorand optionally a gamma-secretase inhibitor. In some cases, thePDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtainedby contacting PDX1-positive pancreatic progenitor cells under conditionsthat promote cell clustering with at least one growth factor from theFGF family. In some cases, the growth factor from the FGF family is KGF.

In some cases, the PDX1-positive pancreatic progenitor cells areproduced from a population of pluripotent cells. In some cases, thePDX1-positive pancreatic progenitor cells are produced from a populationof iPS cells. In some cases, the PDX1-positive pancreatic progenitorcells are produced from a population of ESC cells. In some cases, thePDX1-positive pancreatic progenitor cells are produced from a populationof definitive endoderm cells. In some cases, the PDX1-positivepancreatic progenitor cells are produced from a population of primitivegut tube cells.

Any growth factor from the FGF family capable of inducing PDX1-positivepancreatic progenitor cells to differentiate into NKX6.1-positivepancreatic progenitor cells (e.g., alone, or with any combination of atleast one SHH pathway inhibitor, a ROCK inhibitor, a growth factor fromthe TGF-β superfamily, and at least one retinoic acid signaling pathwayactivator) can be used in the method provided herein. In some cases, theat least one growth factor from the FGF family comprises keratinocytegrowth factor (KGF). In some cases, the at least one growth factor fromthe FGF family is selected from the group consisting of FGF8B, FGF 10,and FGF21. In some examples, the method comprises contactingPDX1-positive pancreatic progenitor cells with a concentration of agrowth factor from FGF family (e.g., KGF), such as, about 10 ng/mL,about 20 ng/mL, about 50 ng/mL, about 75 ng/mL, about 80 ng/mL, about 90ng/mL, about 95 ng/mL, about 100 ng/mL, about 110 ng/mL, about 120ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL, about 175ng/mL, about 180 ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300ng/mL.

Any SHH pathway inhibitor capable of inducing PDX1-positive pancreaticprogenitor cells to differentiate into NKX6.1-positive pancreaticprogenitor cells (e.g., alone, or with any combination of at least onegrowth factor from the FGF family, at least one retinoic acid signalingpathway activator, ROCK inhibitor, and at least one growth factor fromthe TGF-β superfamily) can be used in the method provided herein. Insome cases, the SHH pathway inhibitor comprises Sant1. In some examples,the method comprises contacting PDX1-positive pancreatic progenitorcells with a concentration of a SHH pathway inhibitor (e.g., Sant1),such as, about 0.001 μM, about 0.002 μM, about 0.005 μM, about 0.01 μM,about 0.02 μM, about 0.03 μM, about 0.05 μM about 0.08 μM, about 0.1 μM,about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about 0.16μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM, about0.21 μM, about 0.22 μM, about 0.23 μM, about 0.24 μM, about 0.25 μM,about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.3μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.6 μM, about0.8 μM, about 1 μM, about 2 μM, or about 5 μM.

Any RA signaling pathway activator capable of inducing PDX1-positivepancreatic progenitor cells to differentiate into NKX6.1-positivepancreatic progenitor cells (e.g., alone, or with any combination of atleast one growth factor from the FGF family, at least one SHH pathwayinhibitor, ROCK inhibitor, and at least one growth factor from the TGF-βsuperfamily) can be used. In some cases, the RA signaling pathwayactivator comprises retinoic acid. In some examples, the methodcomprises contacting PDX1-positive pancreatic progenitor cells with aconcentration of an RA signaling pathway activator (e.g., retinoicacid), such as, about 0.02 μM, about 0.1 μM, about 0.2 μM, about 0.25μM, about 0.3 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.55μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM, about 0.8μM, about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM,about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM,about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM,about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM,about 2.8 μM, about 3 μM, about 3.2 μM, about 3.4 μM, about 3.6 μM,about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM, about 4.6 μM,about 4.8 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM,about 10 μM, about 12 μM, about 14 μM, about 15 μM, about 16 μM, about18 μM, about 20 μM, about 50 μM, or about 100 μM.

Any ROCK inhibitor capable of inducing PDX1-positive pancreaticprogenitor cells to differentiate into NKX6.1-positive pancreaticprogenitor cells (e.g., alone, or with any combination of at least onegrowth factor from the FGF family, at least one SHH pathway inhibitor, aRA signaling pathway activator, and at least one growth factor from theTGF-β superfamily) can be used. In some cases, the ROCK inhibitorcomprises Thiazovivin, Y-27632, Fasudil/HA1077, or 14-1152. In someexamples, the method comprises contacting PDX1-positive pancreaticprogenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632or Thiazovivin), such as, about 0.2 μM, about 0.5 μM, about 0.75 μM,about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM,about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 11μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM,about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM,about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about50 μM, or about 100 μM.

Any activator from the TGF-β superfamily capable of inducingPDX1-positive pancreatic progenitor cells to differentiate intoNKX6.1-positive pancreatic progenitor cells (e.g., alone, or with anycombination of at least one growth factor from the FGF family, at leastone SHH pathway inhibitor, a RA signaling pathway activator, and ROCKinhibitor) can be used. In some cases, the activator from the TGF-βsuperfamily comprises Activin A or GDF8. In some examples, the methodcomprises contacting PDX1-positive pancreatic progenitor cells with aconcentration of a growth factor from TGF-β superfamily (e.g., ActivinA), such as, about 0.1 ng/mL, about 0.2 ng/mL, about 0.3 ng/mL, about0.4 ng/mL, about 0.5 ng/mL, about 0.6 ng/mL, about 0.7 ng/mL, about 0.8ng/mL, about 1 ng/mL, about 1.2 ng/mL, about 1.4 ng/mL, about 1.6 ng/mL,about 1.8 ng/mL, about 2 ng/mL, about 2.2 ng/mL, about 2.4 ng/mL, about2.6 ng/mL, about 2.8 ng/mL, about 3 ng/mL, about 3.2 ng/mL, about 3.4ng/mL, about 3.6 ng/mL, about 3.8 ng/mL, about 4 ng/mL, about 4.2 ng/mL,about 4.4 ng/mL, about 4.6 ng/mL, about 4.8 ng/mL, about 5 ng/mL, about5.2 ng/mL, about 5.4 ng/mL, about 5.6 ng/mL, about 5.8 ng/mL, about 6ng/mL, about 6.2 ng/mL, about 6.4 ng/mL, about 6.6 ng/mL, about 6.8ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL,about 20 ng/mL, about 30 ng/mL, or about 50 ng/mL. In some examples, themethod comprises contacting PDX1-positive pancreatic progenitor cellswith a concentration of a growth factor from TGF-β superfamily (e.g.,Activin A), such as, about 5 ng/mL.

In some cases, the PDX1-positive, NKX6.1-positive pancreatic progenitorcells are obtained by contacting PDX1-positive pancreatic progenitorcells under conditions that promote cell clustering with KGF, Sant1, andRA, for a period of 5 days or 6 days. In some cases, the PDX1-positive,NKX6.1-positive pancreatic progenitor cells are obtained by contactingPDX1-positive pancreatic progenitor cells under conditions that promotecell clustering with KGF, Sant1, RA, thiazovivin, and Activin A, for aperiod of 5 or 6 days. In some cases, the PDX1-positive, NKX6.1-positivepancreatic progenitor cells are obtained by contacting PDX1-positivepancreatic progenitor cells under conditions that promote cellclustering with KGF for a period of 5 days. In some embodiments, thePDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtainedby: a) contacting PDX1-positive pancreatic progenitor cells with KGF,Sant1, RA, thiazovivin, and Activin A, for a period of 3, 4 or 5 days,followed by; b) contacting the cells of a) with PDBU, XXI, KGF, Sant1,RA, thiazovivin, and Activin A for a period of 1, 2 or 3 days.

Insulin Ppositive Endocrine Cells

Aspects of the disclosure involve insulin-positive endocrine cells(e.g., NKX6.1-positive, ISL1-positive cells, or β-like cells).Insulin-positive endocrine cells of use herein can be derived from anysource or generated in accordance with any suitable protocol , In someaspects, NKX6.1-positive pancreatic progenitor cells are differentiatedto insulin-positive endocrine cells (e.g., NKX6.1-positive,ISL1-positive cells, or β-like cells), In some aspects, theinsulin-positive endocrine cells are further differentiated, e.g., byinduction or maturation to SC-β cells.

In some aspects, a method of producing an insulin-positive endocrinecell from an NKX6.1-positive pancreatic progenitor cell comprisescontacting a population of cells (e.g., under conditions that promotecell clustering) comprising NKX6-1-positive pancreatic progenitor cellswith a) a TGF-β signaling pathway inhibitor, and b) a thyroid hormonesignaling pathway activator, to induce the differentiation of at leastone NKX6.1-positive pancreatic progenitor cell in the population into aninsulin-positive endocrine cell, wherein the insulin-positive endocrineceil expresses insulin. In some cases, insulin-positive endocrine cellsexpress PDX1, NKX6.1, ISL1, NKX2.2, Mafb, glis3, Sur1, Kir6.2, Znt8,SLC2A1, SLC2A3 and/or insulin.

Any TGF-β signaling pathway inhibitor capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells todifferentiate into insulin-positive endocrine cells (e.g., alone, or incombination with other β cell-differentiation factors, e.g., a thyroidhormone signaling pathway activator) can be used. In some cases, theTGF-β signaling pathway comprises TGF-β receptor type I kinasesignaling. In some cases, the TGF-β signaling pathway inhibitorcomprises Alk5 inhibitor II.

Any thyroid hormone signaling pathway activator capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells todifferentiate into insulin-positive endocrine cells (e.g., alone, or incombination with other β cell-differentiation factors, e.g., a TGF-βsignaling pathway inhibitor) can be used. In some cases, the thyroidhormone signaling pathway activator comprises triiodothyronine (T3). Insome cases, the thyroid hormone signaling pathway activator comprisesGC-1.

In some cases, the method comprises contacting the population of cells(e.g., NKX6.1-positive pancreatic progenitor cells) with at least oneadditional factor. In some cases, the method comprises contacting thePDX1-positive NKX6.1-positive pancreatic progenitor cells with at leastone of i) a SHH pathway inhibitor, ii) a RA signaling pathway activator,iii) a γ-secretase inhibitor, iv) at least one growth factor from theepidermal growth factor (EGF) family, v) a protein kinase inhibitor, vi)a TGF-β signaling pathway inhibitor, or vii) a thyroid hormone signalingpathway activator. In some embodiments, the method comprises contactingthe population of cells (e.g., NKX6.1-positive pancreatic progenitorcells) with at least one additional factor. In some cases, the methodcomprises contacting the PDX1-positive NKX6.1-positive pancreaticprogenitor cells with at least one of i) a SHH pathway inhibitor, ii) aRA signaling pathway activator, iii) a γ-secretase inhibitor, iv) atleast one growth factor from the epidermal growth factor (EGF) family,v) a protein kinase inhibitor, vi) a TGF-β signaling pathway inhibitor,vii) a thyroid hormone signaling pathway activator, or viii) a PKCactivator.

In some cases, the method comprises contacting the PDX1-positiveNKX6.1-positive pancreatic progenitor cells with at least one of i) aSHH pathway inhibitor, ii) a RA signaling pathway activator, iii) aγ-secretase inhibitor, iv) at least one growth factor from the epidermalgrowth factor (EGF) family, v) at least one bone morphogenetic protein(BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathwayinhibitor, vii) a thyroid hormone signaling pathway activator, viii) aprotein kinase inhibitor, or ix) a ROCK inhibitor.

In some cases, the method comprises contacting the PDX1-positiveNKX6.1-positive pancreatic progenitor cells with at least one of i) aSHH pathway inhibitor, ii) a RA signaling pathway activator, iii) aγ-secretase inhibitor, iv) at least one growth factor from the epidermalgrowth factor (EGF) family, v) at least one bone morphogenetic protein(BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathwayinhibitor, vii) a thyroid hormone signaling pathway activator, viii) anepigenetic modifying compound, ix) a protein kinase inhibitor, or x) aROCK inhibitor.

In some embodiments, in the method of generating the insulin-positiveendocrine cells from the PDX1-positive NKX6.1-postive pancreaticprogenitor cells, some of the differentiation factors are present onlyfor the first 1, 2, 3, 4, or 5 days during the differentiation step. Insome cases, some of the differentiation factors, such as the SHH pathwayinhibitor, the RA signaling pathway activator, the PKC activator, andthe at least one growth factor from the EGF family are removed from theculture medium after the first 1, 2, or 3 days of incubation.

Any γ-secretase inhibitor that is capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells in apopulation into insulin-positive endocrine cells (e.g., alone, or incombination with any of a TGF-β signaling pathway inhibitor and/or athyroid hormone signaling pathway activator). In some cases, theγ-secretase inhibitor comprises XXI. In some cases, the γ-secretaseinhibitor comprises DAPT. In some examples, the method comprisescontacting NKX6.1-positive pancreatic progenitor cells with aconcentration of a γ-secretase inhibitor (e.g., XXI), such as, about0.01 μM, about 0.02 μM, about 0.05 μM, about 0.075 μM, about 0.1 μM,about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM,about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.1 μM,about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM,about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM,about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2.6 μM,about 2.7 μM, about 2.8 μM, about 2.9 μM, about 3 μM, about 3.2 μM,about 3.4 μM, about 3.6 μM, about 3.8 μM, about 4 μM, about 4.2 μM,about 4.4 μM, about 4.6 μM, about 4.8 μM, about 5 μM, about 5.2 μM,about 5.4 μM, about 5.6 μM, about 5.8 μM, about 6 μM, about 6.2 μM,about 6.4 μM, about 6.6 μM, about 6.8 μM, about 7 μM, about 8 μM, about9 μM, about 10 μM, about 20 μM, about 30 μM, or about 50 μM.

Any growth factor from the EGF family capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells in apopulation into insulin-positive endocrine cells (e.g., alone, or incombination with any of a TGF-β signaling pathway inhibitor and/or athyroid hormone signaling pathway activator) can be used. In some cases,the at least one growth factor from the EG F family comprisesbetacellulin. In some cases, at least one growth factor from the EGFfamily comprises EGF. In some examples, the method comprises contactingNKX6.1-positive pancreatic progenitor cells with a concentration of agrowth factor from EGF family (e.g., betacellulin), such as, about 1ng/mL, about 2 ng/mL, about 4 ng/mL, about 6 ng/mL, about 8 ng/mL, about10 ng/mL, about 12 ng/mL, about 14 ng/mL, about 16 ng/mL, about 18ng/mL, about 20 ng/mL, about 22 ng/mL, about 24 ng/mL, about 26 ng/mL,about 28 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 75ng/mL, about 80 ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL,about 150 ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL.

Any RA signaling pathway activator capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells todifferentiate into insulin-positive endocrine cells (e.g., alone, or incombination with any of a TGF-β signaling pathway inhibitor and/or athyroid hormone signaling pathway activator) can be used. In some cases,the RA signaling pathway activator comprises RA. In some examples, themethod comprises contacting NKX6.1-positive pancreatic progenitor cellswith a concentration of an RA signaling pathway activator (e.g.,retinoic acid), such as, about 0.02 μM, about 0.1 μM, about 0.2 μM,about 0.25 μM, about 0.3 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM,about 0.55 μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM,about 0.8 μM, about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM,about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM,about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM,about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2.6 μM,about 2.7 μM, about 2.8 μM, about 3 μM, about 3.2 μM, about 3.4 μM,about 3.6 μM, about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM,about 4.6 μM, about 4.8 μM, about 5 μM, about 5.5 μM, about 6 μM, about6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM,about 9.5 μM, about 10 μM, about 12 μM, about 14 μM, about 15 μM, about16 μM, about 18 μM, about 20 μM, about 50 μM, or about 100 μM.

Any SHH pathway inhibitor capable of inducing the differentiation ofNKX6.1-positive pancreatic progenitor cells to differentiate intoinsulin-positive endocrine cells (e.g., alone, or in combination withany of a TGF-β signaling pathway inhibitor and/or a thyroid hormonesignaling pathway activator) can be used in the method provided herein.In some cases, the SHH pathway inhibitor comprises Sant1. In someexamples, the method comprises contacting NKX6.1-positive pancreaticprogenitor cells with a concentration of a SHH pathway inhibitor (e.g.,Sant1), such as, about 0.001 μM, about 0.002 μM, about 0.005 μM, about0.01 μM, about 0.02 μM, about 0.03 μM, about 0.0504, about 0.08 μM,about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about0.2 μM, about 0.21 μM, about 0.22 μM, about 0.23 μM, about 0.24 μM,about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM.

Any BMP signaling pathway inhibitor capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells todifferentiate into insulin-positive endocrine cells (e.g., alone, or incombination with any of a TGF-β signaling pathway inhibitor and/or athyroid hormone signaling pathway activator) can be used. In some cases,the BMP signaling pathway inhibitor comprises LDN193189 or DMH-1. Insome examples, the method comprises contacting NKX6.1-positivepancreatic progenitor cells with a concentration of BMP signalingpathway inhibitor (e.g., LDN1931189), such as, about 30 nM, about 40 nM,about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about250 nM, about 280 nM, about 300 nM, about 400 nM, about 500 nM, or about1 μM.

Any ROCK inhibitor that is capable of inducing the differentiation ofNKX6.1-positive pancreatic progenitor cells in a population intoinsulin-positive endocrine cells (e.g., alone, or in combination withany of a TGF-β signaling pathway inhibitor and/or a thyroid hormonesignaling pathway activator) can be used. In some cases, the ROCKinhibitor comprises Thiazovivin, Y-27632, Fasudil/HA1077, or H-1152. Insome cases, the ROCK inhibitor comprises Y-27632. In some cases, theROCK inhibitor comprises Thiazovivin. In some examples, the methodcomprises contacting PDX1-positive, NKX6.1-positive pancreaticprogenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632or Thiazovivin), such as, about 0.2 μM, about 0.5 μM, about 0.75 μM,about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM,about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 11μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM,about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM,about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about50 μM, or about 100 μM.

Any epigenetic modifying compound that is capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells in apopulation into insulin-positive endocrine cells (e.g., alone, or incombination with any of a TGF-β signaling pathway inhibitor and/or athyroid hormone signaling pathway activator) can be used. In some cases,the epigenetic modifying compound comprises a histone methyltransferaseinhibitor or a HDAC inhibitor. In some cases, the epigenetic modifyingcompound comprises a histone methyltransferase inhibitor, e.g., DZNep.In some cases, the epigenetic modifying compound comprises a HDACinhibitor, e.g., KD5170. In some examples, the method comprisescontacting PDX1-positive, NKX6.1-positive pancreatic progenitor cellswith a concentration of an epigenetic modifying compound (e.g., DZNep orKD5170), such as, about 0.01 μM, about 0.025 μM, about 0.05 μM, about0.075 μM, about 0.1 μM, about 0.15 μM, about 0.2 μM, about 0.5 μM, about0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM,about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM,about 40 μM, about 50 μM, or about 100 μM.

In some cases, the population of cells is optionally contacted with aprotein kinase inhibitor. In some cases, the population of cells is notcontacted with the protein kinase inhibitor. In some cases, thepopulation of cells is contacted with the protein kinase inhibitor. Anyprotein kinase inhibitor that is capable of inducing the differentiationof NKX6.1-positive pancreatic progenitor cells in a population intoinsulin-positive endocrine cells (e.g., alone, or in combination withany of a TGF-β signaling pathway inhibitor and/or a thyroid hormonesignaling pathway activator). In some cases, the protein kinaseinhibitor comprises staurosporine.

In some cases, the method comprises contacting the population of cells(e.g., NKX6.1-positive pancreatic progenitor cells) with XXI, Alk5i, T3or GC-1, RA, Sant1, and betacellulin for a period of 7 days, to inducethe differentiation of at least one NKX6.1-positive pancreaticprogenitor cell in the population into an insulin-positive endocrinecell, wherein the insulin-positive endocrine cell expresses insulin. Insome cases, the method comprises contacting the population of cells(e.g., NKX6.1-positive pancreatic progenitor cells) with XXI, Alk5i, T3or GC-1, RA, Sant1, betacellulin, and LDN193189 for a period of 7 days,to induce the differentiation of at least one NKX6.1-positive pancreaticprogenitor cell in the population into an insulin-positive endocrinecell, wherein the insulin-positive endocrine cell expresses insulin. Insome embodiments, one or more differentiation factors are added in aportion of the Stage 5, for instance, only the first 1, 2, 3, 4, 5, or 6days of the period of time for Stage 5, or the last 1, 2, 3, 4, 5, or 6days of the period of time for Stage 5. In one example, the cells arecontacted with SHH signaling pathway inhibitor for only the first 2, 3,4, or 5 days during Stage 5, after which the SHH signaling pathwayinhibitor is removed from the culture medium. In another example, thecells are contacted with BMP signaling pathway inhibitor for only thefirst 1, 2, or 3 days during Stage 5, after which the BMP signalingpathway inhibitor is removed from the culture medium.

In some cases, the method comprises culturing the population of cells(e.g., NKX6.1-positive pancreatic progenitor cells) in a BE5 medium, toinduce the differentiation of at least one NKX6.1-positive pancreaticprogenitor cell in the population into an insulin-positive endocrinecell, wherein the insulin-positive endocrine cell expresses insulin.

Aspects of the disclosure involve treatment of cell populationcomprising PDX1-positive, NKX6.1-positive pancreatic progenitor cellswith PKC activator, which can lead to increase in percentage ofpancreatic α cells, increase in percentage of pancreatic δ cells,increase in percentage of pancreatic β cells, reduction in percentage ofEC cells, or any combination thereof, in the cell population ofpancreatic endocrine cells generated according to the method disclosedherein.

In some cases, the method comprises contacting a population of cellscomprising PDX1-positive, NKX6.1-positive pancreatic progenitor cellswith a first composition comprising the PKC activator, a ROCK inhibitor,a growth factor from TGFβ superfamily, a growth factor from FGF family,a RA signaling pathway activator, and a SHH pathway inhibitor, for oneto two days, thereby obtaining a first transformation cell populationcomprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells;and contacting the first transformation cell population comprisingPDX1-positive, NKX6.1-positive pancreatic progenitor cells with a secondcomposition comprising the PKC activator, a TGF-β signaling pathwayinhibitor, a TH signaling pathway activator, and an epigenetic modifyingcompound, for one to two days, thereby obtaining a second transformationcell population comprising NKX6.1-positive, ISL1-positive endocrinecells.

In some cases, the method comprises (1) contacting PDX1-positivepancreatic progenitor cells with i) at least one growth factor from theFGF family, ii) at least one SHH pathway inhibitor, and optionally iii)a low concentration of a RA signaling pathway activator, iv) ROCKinhibitor, and v) at least one growth factor from the TGF-β superfamily,for about two to six days, to induce the differentiation of at leastsome of the PDX1-positive pancreatic progenitor cells intoPDX1-positive, NKX6.1-positive pancreatic progenitor cells; and (2)after (1) contacting the population comprising the PDX1-positive,NKX6.1-positive pancreatic progenitor cells with i) at least one growthfactor from the FGF family, ii) at least one SHH pathway inhibitor, iii)a low concentration of a RA signaling pathway activator, iv) ROCKinhibitor, v) at least one growth factor from the TGF-β superfamily, andvi) a PKC activator, for one to two days, thereby generating a firsttransformation cell population comprising PDX1-positive, NKX6.1-positivepancreatic progenitor cells.

In some cases, the method further comprises: (3) contacting firsttransformation cell population comprising PDX1-positive, NKX6.1-positivepancreatic progenitor cells with i) a SHH pathway inhibitor, ii) a RAsignaling pathway activator, iii) a γ-secretase inhibitor, iv) at leastone growth factor from the epidermal growth factor (EGF) family, v) atleast one bone morphogenetic protein (BMP) signaling pathway inhibitor,vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormonesignaling pathway activator, viii) an epigenetic modifying compound, ix)a protein kinase inhibitor, x) a ROCK inhibitor, and xi) a PKCactivator, for one to two days, thereby generating a secondtransformation cell population; and (4) contacting the secondtransformation cell population with i) a SHH pathway inhibitor, ii) a RAsignaling pathway activator, iii) a γ-secretase inhibitor, iv) at leastone growth factor from the epidermal growth factor (EGF) family, v) atleast one bone morphogenetic protein (BMP) signaling pathway inhibitor,vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormonesignaling pathway activator, viii) an epigenetic modifying compound, ix)a protein kinase inhibitor, and x) a ROCK inhibitor, thereby generatingα cell population comprising NKX6.1-positive, ISL1-positive endocrinecells.

Pancreatic β Cells

Aspects of the disclosure involve generating pancreatic β cells (e.g.,non-native pancreatic β cells). Non-native pancreatic β cells, in somecases, resemble endogenous mature β cells in form and function, butnevertheless are distinct from native β cells.

In some cases, the insulin-positive pancreatic endocrine cells generatedusing the method provided herein can form α cell cluster, alone ortogether with other types of cells, e.g., precursors thereof, e.g., stemcell, definitive endoderm cells, primitive gut tube cell, PDX1-positivepancreatic progenitor cells, or NKX6.1-positive pancreatic progenitorcells.

In some embodiments, any of the cells or populations of cells disclosedherein are in a cell cluster. In some aspects, provided herein are cellclusters that resemble the functions and characteristics of endogenouspancreatic islets. Such cell clusters can mimic the function ofendogenous pancreatic islets in regulating metabolism, e.g., glucosemetabolism in a subject. Thus, the cell clusters can be transplanted toa subject for treating disease resulting from insufficient pancreaticislet function, e.g., diabetes. The terms “cluster” and “aggregate” canbe used interchangeably, and refer to a group of cells that have closecell-to-cell contact, and in some cases, the cells in a cluster can beadhered to one another. A cell cluster comprises a plurality of cells.In some embodiments, a cell cluster comprises at least 10, at least 50,at least 200, at least 500, at least 750, at least 1000, at least 1500,at least 2000, at least 2500, at least 3000, at least 3500, at least4000, at least 4500, at least 5000, at least 6000, at least 7000, atleast 8000, at least 9000, at least 10,000, at least 20,000, at least30,000, or at least 50,000 cells. In some embodiments, a cell clustercomprises between 10-10,000 cells, between 50-10,000, between100-10,000, between 100-10,000, between 1,000-10,000, between 500 and10,000, between 500 and 5,000, between 500 and 2,500, between 500 and2,000, between 1,000 and 100,000, between 1,000 and 50,000, between1,000 and 40,000, between 1,000 and 20,000, between 1,000 and 10,000,between 1,000 and 5,000 and between 1,000 and 3,000 cells. In someembodiments, a cell cluster comprises at least 500 cells. In someembodiments, a cell cluster comprises at least 1,000 cells. In someembodiments, a cell cluster comprises at least 2,000 cells. In someembodiments, a cell cluster comprises at least 5,000 cells. In someembodiments, a cell cluster comprises no more than 100,000, no more than90,000, no more than 80,000, no more than 70,000, no more than 60,000,no more than 50,000, no more than 40,000, no more than 30,000, no morethan 20,000, no more than 10,000, no more than 7,000, no more than5,000, no more than 3,000, no more than 2,000 cells, or no more than1,000 cells.

A cell cluster can be in a size similar to an endogenous pancreaticislet. For example, a cell cluster can have a diameter similar to anendogenous pancreatic islet. A diameter of a cell cluster can refer tothe largest linear distance between two points on the surface of thecell cluster. In some cases, the diameter of a cell cluster is at most300 μm, 200 μm, 150 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, or 40μm. The diameter of a cell cluster can be from about 75 μm to about 250μm. The diameter of a cell cluster can be at most 100 μm.

In some embodiments, a cell cluster is between about 100 and about 250microns in diameter (e.g., about 125, about 140, about 150, about 160,about 170, about 180, about 190, about 200, about 200, about 210, about215, about 220, or about 225, microns in diameter). For example, in someembodiments, the cell cluster is between about 125 and about 225,between about 130 and about 160, between about 170 and about 225,between about 140 and about 200, between about 140 and about 170,between about 160 and about 220, between about 170 and about 215, orbetween about 170 and about 200, microns in diameter.

In some embodiments, a composition, cell or cell population of thepresent disclosure comprises cells having a genomic disruption in atleast one gene sequence. In some embodiments, the genomic disruptionreduces or eliminates expression of a protein encoded by said genesequence. In some embodiments, the at least one gene sequence encodes anMHC-Class I gene. In some embodiments, the MHC-Class I gene encodesbeta-2 microglobulin, HLA-A, HLA-B, or HLA-C. In some embodiments, theat least one gene sequence encodes for CIITA. For example, in someembodiments, the composition or cell population has a genomic disruptionin the beta-2-microglobulin gene. Additional examples of genes andgenomic disruptions thereof are described in more detail inInternational Publication No. WO2020/033879, the relevant content ofwhich is incorporated herein by reference. In some embodiments, thegenomic disruption is induced using a gene editing technology (e.g.,CRISPR Cas).

In some embodiments, a composition or cell population of the presentdisclosure comprises NKX6.1-positive, ISL-positive cells that expresslower levels of MAFA than NKX6.1-positive, ISL-positive cells from thepancreas of a healthy control adult subject. In some embodiments, thecomposition or cell population comprises NKX6.1-positive, ISL-positivecells that express higher levels of MAFB than NKX6.1-positive,ISL-positive cells from the pancreas of a healthy control adult subject.In some embodiments, the composition or cell population comprisesNKX6.1-positive, ISL-positive cells that express higher levels of SIX2,HOPX, IAPP and/or UCN3 than NKX6.1-positive, ISL-positive cells from thepancreas of a healthy control adult subject.

In some embodiments, a composition or cell population of the presentdisclosure comprises NKX6.1-positive, ISL-positive cells that do notexpress MAFA. In some embodiments, the composition or cell populationcomprises NKX6.1-positive, ISL-positive cells that express MAFB.

In some cases, the cell population comprising the insulin-positiveendocrine cells can be directly induced to mature into SC-β cellswithout addition of any exogenous differentiation factors (such asinhibitor of TGF-β signaling pathway, thyroid hormone signaling pathwayactivator, PKC activator, growth factors from TGF-β superfamily, FGFfamily, or EGF family, SHH signaling pathway inhibitor, γ-secretaseinhibitor, ROCK inhibitor, or BMP signaling pathway inhibitor). In someembodiments, the method provided herein comprises contacting a cellpopulation comprising NKX6.1-positive, ISL1-positive endocrine cellswith a serum albumin protein, a TGF-β signaling pathway inhibitor, a SHHpathway inhibitor, a TH signaling pathway activator, a protein kinaseinhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and/oran epigenetic modifying compound. In some embodiments, the methodprovided herein comprises contacting a cell population comprisingNKX6.1-positive, ISL1-positive endocrine cells with human serum albuminprotein. In some embodiments, the method provided herein comprisescontacting a cell population comprising NKX6.1-positive, ISL1-positiveendocrine cells with a PKC activator.

In some cases, the cell population comprising the insulin-positiveendocrine cells can be induced to mature into SC-β cells by contactingthe insulin-positive endocrine cells with differentiation factors. Thedifferentiation factors can comprise at least one inhibitor of TGF-βsignaling pathway and thyroid hormone signaling pathway activator asdescribed herein. In some cases, SC-β cells can be obtained bycontacting a population of cells comprising insulin-positive endocrinecells with Alk5i and T3 or GC-1.

In some cases, the method provided herein comprises contacting a cellpopulation comprising NKX6.1-positive, ISL1-positive endocrine cellswith (i) a growth factor from the FGF family, (ii) a TGF-β signalingpathway inhibitor, (iii) a thyroid hormone signaling pathway activator,(iv) an epigenetic modifying compound, (v) a protein kinase inhibitor,(vi) a ROCK inhibitor, (vii) a BMP signaling pathway inhibitor, and(viii) a lipase inhibitor for about one two five days. In some cases,the contacting is for about three days.

In some examples, insulin-positive endocrine cells can be matured in aNS-GFs medium, MCDB131 medium, DMEM medium, or CMRL medium. In somecases, the insulin-positive endocrine cells can be matured in a CMRLsmedium supplemented with 10% FBS. In some cases, the insulin-positiveendocrine cells can be matured in a DMEM/F12 medium supplemented with 1%HSA. In other cases, SC-β cells can be obtained by culturing thepopulation of cells containing the insulin-positive endocrine cells in aMCDB131 medium that can be supplemented by 2% BSA. In some cases, theMCDB131 medium with 2% BSA for maturation of insulin-positive endocrinecells into SC-β cells can be comprise no small molecule factors asdescribed herein. In some case, the MCDB131 medium with 2% BSA formaturation of insulin-positive endocrine cells into SC-β cells cancomprise no serum (e.g., no FBS). In other cases, SC-β cells can beobtained by culturing the population of cells containing theinsulin-positive endocrine cells in a MCDB131 medium that can besupplemented by 0.05% HSA and vitamin C. In some cases, SC-β cells canbe obtained by culturing the population of cells containing theinsulin-positive endocrine cells in a MCDB131 medium that can besupplemented by 0.05% HSA, ITS-X, vitamin C, and glutamine (Gln, e.g., 4mM). In some cases, the type of culture medium may be changed during S6.For instance, the S6 cells are cultured in a MCDB131 medium that can besupplemented by 0.05% HSA and vitamin C for the first two to four days,and then followed by a DMEM/F12 medium supplemented with 1% HSA. In somecases, additional factors are introduced into the culture medium. Forinstance, S6 cells can be cultured in a MCDB131 medium that can besupplemented by 0.05% HSA, ITS-X, vitamin C, and glutamine (Gln, e.g., 4mM) throughout the 10-12 days, during which ZnSO₄ is introduced from day4 of S6.

In some aspects, the disclosure provides a method of generating SC-βcells from pluripotent cells, the method comprising: a) differentiatingpluripotent stem cells in a population into definitive endoderm cells bycontacting the pluripotent stem cells with at least one factor from TGFβsuperfamily and a WNT signaling pathway activator for a period of 3days; b) differentiating at least some of the definitive endoderm cellsinto primitive gut tube cells by a process of contacting the definitiveendoderm cells with at least one factor from the FGF family for a periodof 3 days; c) differentiating at least some of the primitive gut tubecells into PDX1-positive pancreatic progenitor cells by a process ofcontacting the primitive gut tube cells with i)retinoic acid signalingpathway activator, ii) at least one factor from the FGF family, iii) aSHH pathway inhibitor, iv) a BMP signaling pathway inhibitor (e.g.,DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor; d)differentiating at least some of the PDX1-positive pancreatic progenitorcells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells bya process of contacting the PDX1-positive pancreatic progenitor cellsunder conditions that promote cell clustering with i) at least onegrowth factor from the FGF family, ii) at least one SHH pathwayinhibitor, and optionally iii) a RA signaling pathway activator, andoptionally iv) ROCK inhibitor and v) at least one factor from TGFβuperfamily, for a period of 5 days ; e) differentiating at least some ofthe PDX1-positive, NKX6.1-positive pancreatic progenitor cells intoPDX1-positive, NKX6.1-positive, insulin-positive endocrine cells by aprocess of contacting the PDX1-positive, NKX6.1-positive pancreaticprogenitor cells with i) a TGF-β signaling pathway inhibitor, ii) a THsignaling pathway activator, iii) at least one SHH pathway inhibitor,iv) a RA signaling pathway activator, v) a γ-secretase inhibitor,optionally vi) at least one growth factor from the epidermal growthfactor (EGF) family, and optionally vii) a BMP signaling pathwayinhibitor, for a period of between five and seven days; and f)differentiating at least some of the PDX1-positive, NKX6.1-positive,insulin-positive endocrine cells into SC-β cells by a process ofculturing the PDX1-positive, NKX6.1-positive, insulin-positive endocrinecells in a medium (e.g., NS-GFs medium, MCDB medium supplemented withBSA, MCDB131 medium, or DMEM/F12 medium)without exogenousdifferentiation factors, for a period of between 7 and 14 days to inducethe in vitro maturation of at least some of the PDX1-positive,NKX6.1-positive, insulin-positive endocrine cells into SC-β cells,wherein the SC-β cells exhibit a GSIS response in vitro and/or in vivo.In some cases, the GSIS response resembles the GSIS response of anendogenous mature β cells.

In some aspects, the disclosure provides a method of generating SC-βcells from pluripotent cells, the method comprising: a) differentiatingpluripotent stem cells in a population into definitive endoderm cells bycontacting the pluripotent stem cells with at least one factor from TGFβuperfamily and a WNT signaling pathway activator for a period of 3 days;b) differentiating at least some of the definitive endoderm cells intoprimitive gut tube cells by a process of contacting the definitiveendoderm cells with at least one factor from the FGF family for a periodof 3 days; c) differentiating at least some of the primitive gut tubecells into PDX1-positive pancreatic progenitor cells by a process ofcontacting the primitive gut tube cells with i) retinoic acid signalingpathway activator, ii) at least one factor from the FGF family, iii) aSHH pathway inhibitor, iv) a BMP signaling pathway inhibitor, v) a PKCactivator, vi) a ROCK inhibitor, and vii) a growth factor from TGFβuperfamily, for a period of 2 days; d) differentiating at least some ofthe PDX1-positive pancreatic progenitor cells into PDX1-positive,NKX6.1-positive pancreatic progenitor cells by a process of contactingthe PDX1-positive pancreatic progenitor cells under conditions thatpromote cell clustering with i) at least one growth factor from the FGFfamily, ii) at least one SHH pathway inhibitor, and optionally iii) a RAsignaling pathway activator, and optionally iv) ROCK inhibitor and v) atleast one factor from TGFβ uperfamily, for a period of 5 days; e)differentiating at least some of the PDX1-positive, NKX6.1-positivepancreatic progenitor cells into PDX1-positive, NKX6.1-positive,insulin-positive endocrine cells by a process of contacting thePDX1-positive, NKX6.1-positive pancreatic progenitor cells with i) aTGF-β signaling pathway inhibitor, ii) a TH signaling pathway activator,iii) at least one SHH pathway inhibitor, iv) a RA signaling pathwayactivator, v) a γ-secretase inhibitor, optionally vi) at least onegrowth factor from the epidermal growth factor (EGF) family, andoptionally vii) a BMP signaling pathway inhibitor, for a period ofbetween five and seven days; and f) differentiating at least some of thePDX1-positive, NKX6.1-positive, insulin-positive endocrine cells intoSC-β cells by a process of culturing the PDX1-positive, NKX6.1-positive,insulin-positive endocrine cells in a medium (e.g., NS-GFs medium, MCDBmedium supplemented with BSA, MCDB131 medium, or DMEM/F12 medium)withoutexogenous differentiation factors, for a period of between 7 and 14 daysto induce the in vitro maturation of at least some of the PDX1-positive,NKX6.1-positive, insulin-positive endocrine cells into SC-β cells,wherein the SC-β cells exhibit a GSIS response in vitro and/or in vivo.In some cases, the GSIS response resembles the GSIS response of anendogenous mature β cells.

In some aspects, the disclosure provides a method of generating SC-βcells from pluripotent cells, the method comprising: a) differentiatingpluripotent stem cells in a population into definitive endoderm cells bycontacting the pluripotent stem cells with at least one factor from TGFβuperfamily and a WNT signaling pathway activator for a period of 3 days;b) differentiating at least some of the definitive endoderm cells intoprimitive gut tube cells by a process of contacting the definitiveendoderm cells with at least one factor from the FGF family for a periodof 3 days; c) differentiating at least some of the primitive gut tubecells into PDX1-positive pancreatic progenitor cells by a process ofcontacting the primitive gut tube cells with i)retinoic acid signalingpathway activator, ii) at least one factor from the FGF family, iii) aSHH pathway inhibitor, iv) a PKC activator, and v) a ROCK inhibitor; d)differentiating at least some of the PDX1-positive pancreatic progenitorcells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells bya process of contacting the PDX1-positive pancreatic progenitor cellsunder conditions that promote cell clustering with i) at least onegrowth factor from the FGF family, ii) at least one SHH pathwayinhibitor, and optionally iii) a RA signaling pathway activator, andoptionally iv) ROCK inhibitor and v) at least one factor from TGFβuperfamily, for a period of 5 days; e) differentiating at least some ofthe PDX1-positive, NKX6.1-positive pancreatic progenitor cells intoPDX1-positive, NKX6.1-positive, insulin-positive endocrine cells by aprocess of contacting the PDX1-positive, NKX6.1-positive pancreaticprogenitor cells with i) a TGF-β signaling pathway inhibitor, ii) a THsignaling pathway activator, iii) at least one SHH pathway inhibitor,iv) a RA signaling pathway activator, v) a γ-secretase inhibitor, andoptionally vi) at least one growth factor from the epidermal growthfactor (EGF) family, for a period of between five and seven days; and f)differentiating at least some of the PDX1-positive, NKX6.1-positive,insulin-positive endocrine cells into SC-β cells by a process ofculturing the PDX1-positive, NKX6.1-positive, insulin-positive endocrinecells in a medium (e.g., NS-GFs medium, MCDB medium supplemented withBSA, MCDB131 medium, or DMEM/F12 medium)without exogenousdifferentiation factors, for a period of between 7 and 14 days to inducethe in vitro maturation of at least some of the PDX1-positive,NKX6.1-positive, insulin-positive endocrine cells into SC-β cells,wherein the SC-β cells exhibit a GSIS response in vitro and/or in vivo.In some cases, the GSIS response resembles the GSIS response of anendogenous mature β cells.

In some aspects, the disclosure provides a method of generating SC-βcells from pluripotent cells, the method comprising: a) differentiatingpluripotent stem cells in a population into definitive endoderm cells bycontacting the pluripotent stem cells with at least one factor from TGFβuperfamily and a WNT signaling pathway activator for a period of 3 days;b) differentiating at least some of the definitive endoderm cells intoprimitive gut tube cells by a process of contacting the definitiveendoderm cells with at least one factor from the FGF family for a periodof 3 days; c) differentiating at least some of the primitive gut tubecells into PDX1-positive pancreatic progenitor cells by a process ofcontacting the primitive gut tube cells with i)retinoic acid signalingpathway activator, ii) at least one factor from the FGF family, iii) aSHH pathway inhibitor, iv) a BMP signaling pathway inhibitor (e.g.,DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor; d)differentiating at least some of the PDX1-positive pancreatic progenitorcells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells bya process of contacting the PDX1-positive pancreatic progenitor cellsunder conditions that promote cell clustering with i) at least onegrowth factor from the FGF family, ii) at least one SHH pathwayinhibitor, and optionally iii) a RA signaling pathway activator, andoptionally iv) ROCK inhibitor and v) at least one factor from TGFβuperfamily, for a period of 5 or 6 days ; e) differentiating at leastsome of the PDX1-positive, NKX6.1-positive pancreatic progenitor cellsinto PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells bya process of contacting the PDX1-positive, NKX6.1-positive pancreaticprogenitor cells with i) a SHH pathway inhibitor, ii) a RA signalingpathway activator, iii) a γ-secretase inhibitor, iv) at least one growthfactor from the epidermal growth factor (EGF) family, v) at least onebone morphogenetic protein (BMP) signaling pathway inhibitor, vi) aTGF-β signaling pathway inhibitor, vii) a thyroid hormone signalingpathway activator, viii) an epigenetic modifying compound (e.g., DZNepor KD5170), ix) a protein kinase inhibitor, and x) a ROCK inhibitor, fora period of between five and seven days; and f) differentiating at leastsome of the PDX1-positive, NKX6.1-positive, insulin-positive endocrinecells into SC-β cells by a process of culturing the PDX1-positive,NKX6.1-positive, insulin-positive endocrine cells in a medium (e.g.,NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, orDMEM/F12 medium) without exogenous differentiation factors, for a periodof between 7 and 14 days to induce the in vitro maturation of at leastsome of the PDX1-positive, NKX6.1-positive, insulin-positive endocrinecells into SC-β cells, wherein the SC-β cells exhibit a GSIS response invitro and/or in vivo. In some cases, the GSIS response resembles theGSIS response of an endogenous mature β cells.

In some aspects, the disclosure provides a method of generating SC-βcells from pluripotent cells, the method comprising: a) differentiatingpluripotent stem cells in a population into definitive endoderm cells bycontacting the pluripotent stem cells with at least one factor from TGFβuperfamily and a WNT signaling pathway activator for a period of 3 days;b) differentiating at least some of the definitive endoderm cells intoprimitive gut tube cells by a process of contacting the definitiveendoderm cells with at least one factor from the FGF family for a periodof 3 days; c) differentiating at least some of the primitive gut tubecells into PDX1-positive pancreatic progenitor cells by a process ofcontacting the primitive gut tube cells with i)retinoic acid signalingpathway activator, ii) at least one factor from the FGF family, iii) aSHH pathway inhibitor, iv) a BMP signaling pathway inhibitor (e.g.,DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor; d)differentiating at least some of the PDX1-positive pancreatic progenitorcells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells bya process of contacting the PDX1-positive pancreatic progenitor cellsunder conditions that promote cell clustering with i) at least onegrowth factor from the FGF family, ii) at least one SHH pathwayinhibitor, and optionally iii) a RA signaling pathway activator, andoptionally iv) ROCK inhibitor and v) at least one factor from TGFβuperfamily, for a period of 5 or 6 days; e) differentiating at leastsome of the PDX1-positive, NKX6.1-positive pancreatic progenitor cellsinto PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells bya process of contacting the PDX1-positive, NKX6.1-positive pancreaticprogenitor cells with i) a γ-secretase inhibitor, ii) at least one bonemorphogenetic protein (BMP) signaling pathway inhibitor, iii) a TGF-βsignaling pathway inhibitor, iv) a thyroid hormone signaling pathwayactivator, v) an epigenetic modifying compound (e.g., DZNep or KD5170),vi) a protein kinase inhibitor, and vii) a ROCK inhibitor, for a periodof between five and seven days, and within first three days of theperiod of between five and seven days, contacting the PDX1-positive,NKX6.1-positive pancreatic progenitor cells with a SHH pathwayinhibitor, a RA signaling pathway, and at least one growth factor fromthe EGF family, which are removed from the PDX1-positive,NKX6.1-positive pancreatic progenitor cells thereafter; and f)differentiating at least some of the PDX1-positive, NKX6.1-positive,insulin-positive endocrine cells into SC-β cells by a process ofculturing the PDX1-positive, NKX6.1-positive, insulin-positive endocrinecells in a medium (e.g., NS-GFs medium, MCDB medium supplemented withBSA, MCDB131 medium, or DMEM/F12 medium) without exogenousdifferentiation factors, for a period of between 7 and 14 days to inducethe in vitro maturation of at least some of the PDX1-positive,NKX6.1-positive, insulin-positive endocrine cells into SC-β cells,wherein the SC-β cells exhibit a GSIS response in vitro and/or in vivo.In some cases, the GSIS response resembles the GSIS response of anendogenous mature β cells.

In some aspects, the disclosure provides a method of generating SC-βcells from pluripotent cells, the method comprising: a) differentiatingpluripotent stem cells in a population into definitive endoderm cells bycontacting the pluripotent stem cells with at least one factor from TGFβuperfamily and a WNT signaling pathway activator for a period of 3 days;b) differentiating at least some of the definitive endoderm cells intoprimitive gut tube cells by a process of contacting the definitiveendoderm cells with at least one factor from the FGF family for a periodof 3 days; c) differentiating at least some of the primitive gut tubecells into PDX1-positive pancreatic progenitor cells by a process ofcontacting the primitive gut tube cells with i)retinoic acid signalingpathway activator, ii) at least one factor from the FGF family, iii) aSHH pathway inhibitor, iv) a BMP signaling pathway inhibitor (e.g.,DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor; d)differentiating at least some of the PDX1-positive pancreatic progenitorcells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells bya process of contacting the PDX1-positive pancreatic progenitor cellsunder conditions that promote cell clustering with i) at least onegrowth factor from the FGF family, ii) at least one SHH pathwayinhibitor, and optionally iii) a RA signaling pathway activator, andoptionally iv) ROCK inhibitor and v) at least one factor from TGFβuperfamily, for a period of 3 or 4 days, followed by contacting with i)at least one growth factor from the FGF family, ii) at least one SHHpathway inhibitor, and optionally iii) a RA signaling pathway activator,and optionally iv) ROCK inhibitor, v) at least one factor from TGFβuperfamily, and vi) a PKC activator, and optionally vii) a gammasecretase inhibitor, for 1 to 2 days; e) differentiating at least someof the PDX1-positive, NKX6.1-positive pancreatic progenitor cells intoPDX1-positive, NKX6.1-positive, insulin-positive endocrine cells by aprocess of contacting the PDX1-positive, NKX6.1-positive pancreaticprogenitor cells with i) a SHH pathway inhibitor, ii) a RA signalingpathway activator, iii) a γ-secretase inhibitor, iv) at least one growthfactor from the epidermal growth factor (EGF) family, v) at least onebone morphogenetic protein (BMP) signaling pathway inhibitor, vi) aTGF-β signaling pathway inhibitor, vii) a thyroid hormone signalingpathway activator, viii) an epigenetic modifying compound (e.g., DZNepor KD5170), ix) a protein kinase inhibitor, x) a ROCK inhibitor, and xi)a PKC activator, for 1 to 2 days, followed by contacting with i) a SHHpathway inhibitor, ii) a RA signaling pathway activator, iii) aγ-secretase inhibitor, iv) at least one growth factor from the epidermalgrowth factor (EGF) family, v) at least one bone morphogenetic protein(BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathwayinhibitor, vii) a thyroid hormone signaling pathway activator, viii) anepigenetic modifying compound (e.g., DZNep or KD5170), ix) a proteinkinase inhibitor, and x) a ROCK inhibitor, for a period of between threeand six days; and f) differentiating at least some of the PDX1-positive,NKX6.1-positive, insulin-positive endocrine cells into SC-β cells.

The medium used to culture the cells dissociated from the first cellcluster can be xeno-free. A xeno-free medium for culturing cells and/orcell clusters of originated from an animal can have no product fromother animals. In some cases, a xeno-free medium for culturing humancells and/or cell clusters can have no products from any non-humananimals. For example, a xeno-free medium for culturing human cellsand/or cell clusters can comprise human platelet lysate (PLT) instead offetal bovine serum (FBS). For example, a medium can comprise from about1% to about 20%, from about 5% to about 15%, from about 8% to about 12%,from about 9 to about 11% serum. In some cases, medium can compriseabout 10% of serum. In some cases, the medium can be free of smallmolecules and/or FBS. For example, a medium can comprise MCDB131 basalmedium supplemented with 2% BSA. In some cases, the medium isserum-free. In some examples, a medium can comprise no exogenous smallmolecules or signaling pathway agonists or antagonists, such as, growthfactor from fibroblast growth factor family (FGF, such as FGF2, FGF8B,FGF 10, or FGF21), Sonic Hedgehog Antagonist (such as Sant1, Sant2,Sant4, Sant4, Cur61414, forskolin, tomatidine, AY9944, triparanol,cyclopamine, or derivatives thereof), Retinoic Acid Signaling agonist(e.g., retinoic acid, CD1530, AM580, TTHPB, CD437, Ch55, BMS961,AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, or CD2314),inhibitor of Rho-associated, coiled-coil containing protein kinase(ROCK) (e.g., Thiazovivin, Y-27632, Fasudil/HA1077, or 14-1152),activator of protein kinase C (PKC) (e.g., phorbol 12,13-dibutyrate(PDBU) , TPB, phorbol 12-myristate 13-acetate, bryostatin 1, orderivatives thereof), antagonist of TGF β super family (e.g., Alk5inhibitor II (CAS 446859-33-2), A83-01, SB431542, D4476, GW788388,LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, SB-505124, orderivatives thereof), inhibitor of Bone Morphogenetic Protein (BMP) type1 receptor (e.g., LDN193189 or derivatives thereof), thyroid hormonesignaling pathway activator (e.g., T3, GC-1 or derivatives thereof),gamma-secretase inhibitor (e.g., XXI, DAPT, or derivatives thereof),activator of TGF-β signaling pathway (e.g., WNT3a or Activin A) growthfactor from epidermal growth factor (EGF) family (e.g., betacellulin orEGF), broad kinase (e.g., staurosporine or derivatives thereof),non-essential amino acids, vitamins or antioxidants (e.g., cyclopamine,vitamin D, vitamin C, vitamin A, or derivatives thereof), or otheradditions like N-acetyl cysteine, zinc sulfate, or heparin. In somecases, the reaggregation medium can comprise no exogenous extracellularmatrix molecule. In some cases, the reaggregation medium does notcomprise Matrigel™. In some cases, the reaggregation medium does notcomprise other extracellular matrix molecules or materials, such as,collagen, gelatin, poly-L-lysine, poly-D-lysine, vitronectin, laminin,fibronectin, PLO laminin, fibrin, thrombin, and RetroNectin and mixturesthereof, for example, or lysed cell membrane preparations.

A person of ordinary skill in the art will appreciate that that theconcentration of serum albumin supplemented into the medium may vary.For example, a medium (e.g., MCDB131) can comprise about 0.01%, 0.05%,0.1%, 1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about15% BSA. In other cases, a medium can comprise about 0.01%, 0.05%, 0.1%,1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about 15% HSA.The medium used (e.g., MCDB131 medium) can contain components not foundin traditional basal media, such as trace elements, putrescine, adenine,thymidine, and higher levels of some amino acids and vitamins. Theseadditions can allow the medium to be supplemented with very low levelsof serum or defined components. The medium can be free of proteinsand/or growth factors, and may be supplemented with EGF, hydrocortisone,and/or glutamine. The medium can comprise one or more extracellularmatrix molecules (e.g., extracellular proteins). Non-limiting exemplaryextracellular matrix molecules used in the medium can include collagen,placental matrix, fibronectin, laminin, merosin, tenascin, heparin,heparin sulfate, chondroitin sulfate, dermatan sulfate, aggrecan,biglycan, thrombospondin, vitronectin, and decorin. In some cases, themedium comprises laminin, such as LN-332. In some cases, the mediumcomprises heparin.

The medium can be changed periodically in the culture, e.g., to provideoptimal environment for the cells in the medium. When culturing thecells dissociated from the first cell cluster for re-aggregation, themedium can be changed at least or about every 4 hours, 12 hours, 24hours, 48 hours, 3 days or 4 days. For example, the medium can bechanged about every 48 hours.

In some cases, cells can be cultured under dynamic conditions (e.g.,under conditions in which the cells are subject to constant movement orstirring while in the suspension culture). For dynamic culturing ofcells, the cells can be cultured in a container (e.g., an non-adhesivecontainer such as a spinner flask (e.g., of 200 ml to 3000 ml, forexample 250 ml; of 100 ml; or in 125 ml Erlenmeyer), which can beconnected to a control unit and thus present a controlled culturingsystem. In some cases, cells can be cultured under non-dynamicconditions (e.g., a static culture) while preserving their proliferativecapacity. For non-dynamic culturing of cells, the cells can be culturedin an adherent culture vessel. An adhesive culture vessel can be coatedwith any of substrates for cell adhesion such as extracellular matrix(ECM) to improve the adhesiveness of the vessel surface to the cells.The substrate for cell adhesion can be any material intended to attachstem cells or feeder cells (if used). The substrate for cell adhesionincludes collagen, gelatin, poly-L-lysine, poly-D-lysine, vitronectin,laminin, fibronectin, PLO laminin, fibrin, thrombin, and RetroNectin andmixtures thereof, for example, Matrigel™, and lysed cell membranepreparations.

Medium in a dynamic cell culture vessel (e.g., a spinner flask) can bestirred (e.g., by a stirrer). The spinning speed can correlate with thesize of the re-aggregated second cell cluster. The spinning speed can becontrolled so that the size of the second cell cluster can be similar toan endogenous pancreatic islet. In some cases, the spinning speed iscontrolled so that the size of the second cell cluster can be from about75 μm to about 250 μm. The spinning speed of a dynamic cell culturevessel (e.g., a spinner flask) can be about 20 rounds per minute (rpm)to about 100 rpm, e.g., from about 30 rpm to about 90 rpm, from about 40rpm to about 60 rpm, from about 45 rpm to about 50 rpm. In some cases,the spinning speed can be about 50 rpm.

Stage 6 cells as provided herein may or may not be subject to thedissociation and reaggregation process as described herein. In somecases, the cell cluster comprising the insulin-positive endocrine cellscan be reaggregated. The reaggregation of the cell cluster can enrichthe insulin-positive endocrine cells. In some cases, theinsulin-positive endocrine cells in the cell cluster can be furthermatured into pancreatic β cells. For example, after reaggregation, thesecond cell cluster can exhibit in vitro GSIS, resembling nativepancreatic islet. For example, after reaggregation, the second cellcluster can comprise non-native pancreatic β cell that exhibits in vitroGSIS. In some embodiments, the reaggregation process can be performedaccording to the disclosure of PCT application PCT/US2018/043179, whichis incorporated herein by reference in its entirety.

Stage 6 cells obtained according to methods provided herein can havehigh recovery yield after cryopreservation and reaggregation procedures.In some cases, stage 6 cells that are obtained in a differentiationprocess that involves treatment of a BMP signaling pathway inhibitor(e.g., DMH-1 or LDN) and a growth factor from TGF-β superfamily (e.g.,Activin A) at stage 3 and treatment of an epigenetic modifying compound(e.g., histone methyltransferase inhibitor, e.g., EZH2 inhibitor, e.g.,DZNep) at stage 5 can have a higher recovery yield aftercryopreservation post stage 5, as compared to a corresponding cellpopulation without such treatment. In some cases, stage 6 cells that areobtained in a differentiation process that involves treatment of a BMPsignaling pathway inhibitor (e.g., DMH-1 or LDN) and a growth factorfrom TGF-β superfamily (e.g., Activin A) at stage 3 and treatment of anepigenetic modifying compound (e.g., histone methyltransferaseinhibitor, e.g., EZH2 inhibitor, e.g., DZNep) at stage 5 can have ahigher recovery yield after cryopreservation post stage 5, as comparedto a corresponding cell population without treatment of a BMP signalingpathway inhibitor (e.g., DMH-1 or LDN) and a growth factor from TGF-βsuperfamily (e.g., Activin A) at stage 3. In some cases, stage 6 cellsthat are obtained in a differentiation process that involves treatmentof a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN) and a growthfactor from TGF-β superfamily (e.g., Activin A) at stage 3 and treatmentof an epigenetic modifying compound (e.g., histone methyltransferaseinhibitor, e.g., EZH2 inhibitor, e.g., DZNep) at stage 5 can have arecovery yield after cryopreservation post stage 5 that is at leastabout 35%, 37.5%, 40%, 42.5%, 45%, 47.5%, 48%, 49%, or 50%. The recoveryyield can be calculated as a percentage of cells that survive and formreaggregated cell clusters after cryopreservation, thawing and recovery,and reaggregation procedures, as compared to the cells before thecryopreservation.

In some embodiments, the present disclosure relates to cryopreservationof the non-native pancreatic β cells or precursors thereof obtainedusing the methods provided herein. In some embodiments, the cellpopulation comprising non-native pancreatic β cells can be stored viacryopreservation. For instances, the cell population comprisingnon-native β cells, e.g., Stage 6 cells in some cases, can bedissociated into cell suspension, e.g., single cell suspension, and thecell suspension can be cryopreserved, e.g., frozen in a cryopreservationsolution. The dissociation of the cells can be conducted by any of thetechnique provided herein, for example, by enzymatic treatment. Thecells can be frozen at a temperature of at highest −20° C., at highest−30° C., at highest −40° C., at highest −50° C., at highest −60° C., athighest −70° C., at highest −80° C., at highest −90° C., at highest−100° C., at highest −110° C., at highest −120° C., at highest −130° C.,at highest −140° C., at highest −150° C., at highest −160° C., athighest −170° C., at highest −180° C., at highest −190° C., or athighest −200° C. In some cases, the cells are frozen at a temperature ofabout −80° C. In some cases, the cells are frozen at a temperature ofabout −195° C. Any cooling methods can be used for providing the lowtemperature needed for cryopreservation, such as, but not limited to,electric freezer, solid carbon dioxide, and liquid nitrogen. In somecases, any cryopreservation solution available to one skilled in the artcan be used for incubating the cells for storage at low temperature,including both custom made and commercial solutions. For example, asolution containing a cryoprotectant can be used. The cryoprotectant canbe an agent that is configured to protect the cell from freezing damage.For instance, a cryoprotectant can be a substance that can lower theglass transition temperature of the cryopreservation solution. Exemplarycryoprotectants that can be used include DMSO (dimethyl sulfoxide),glycols (e.g., ethylene glycol, propylene glycol and glycerol), dextran(e.g., dextran-40), and trehalose. Additional agents can be added in tothe cryopreservation solution for other effects. In some cases,commercially available cryopreservation solutions can be used in themethod provided herein, for instance, FrostaLife™, pZerve™, Prime-XV®,Gibco Synth-a-Freeze Cryopreservation Medium, STEM-CELLBANKER200 ,CryoStor® Freezing Media, HypoThermosol® FRS Preservation Media, andCryoDefend® Stem Cells Media.

During the differentiation process, the cells can be subject toirradiation treatment as provided herein. In some cases, the cellpopulation at Stage 6, e.g., the cell population or cell cluster thathas cells being differentiated from insulin-positive endocrine cellsinto pancreatic β cells, is irradiated for a period of time. In somecases, the cell population at Stage 6 after reaggregation following therecovery from cryopreservation is irradiated for a period of time. Insome cases, the cryopreserved cells (e.g., the cells that arecryopreserved at the end of Stage 5) are irradiated for a certain periodof time prior to thawing and recovery for subsequent differentiationprocess.

In some embodiments, the stage 6 cells comprise NKX6.1-positive,insulin-positive cells. In some embodiments, the stage 6 cells compriseNKX6.1-positive, insulin-negative cells. In some embodiments, the stage6 cells comprise C-peptide positive cells. In some embodiments, Stage 6cells or cells that have characteristics of stage 6 cells are incubatedin NS-GFs medium, MCDB131 medium, DMEM medium, or CMRL medium. In someembodiments, the stage 6 cells or cells that have characteristics ofstage 6 cells are contacted with any one or more of a vitamin oranti-oxidant (e.g., vitamin C), an albumin protein (e.g., a human serumalbumin protein), a TGF-beta pathway inhibitor (e.g., an ALKS inhibitorII), a bone morphogenic protein (BMP) type 1 receptor inhibitor (e.g.,LDN193189), a Rho-associated coiled-coil containing protein kinase(ROCK) inhibitor (e.g., thiazovivin), a histone methyltransferaseinhibitor (e.g., DZNEP), and a protein kinase inhibitor (e.g.,staurosporine). In some embodiments, the stage 6 cells are contactedwith a PKC activator (see, e.g., WO2019217487, which is incorporated byreference herein in its entirety).

Differentiation Factors

Aspects of the disclosure relate to contacting progenitor cells (e.g.,stem cells, e.g., iPS cells, definitive endoderm cells, primitive guttube cells, PDX1-positive pancreatic progenitor cells, NKX6.1-positivepancreatic progenitor cells, insulin-positive endocrine cells) with βcell differentiation factors, for example, to induce the maturation ofthe insulin-positive endocrine cells or differentiation of otherprogenitor cells into SC-β cells (e.g., mature pancreatic β cells). Insome embodiments, the differentiation factor can induce thedifferentiation of pluripotent cells (e.g., iPSCs or hESCs) intodefinitive endoderm cells, e.g., in accordance with a method describedherein. In some embodiments, the differentiation factor can induce thedifferentiation of definitive endoderm cells into primitive gut tubecells, e.g., in accordance with a method described herein. In someembodiments, the differentiation factor can induce the differentiationof primitive gut tube cells into PDX1-positive pancreatic progenitorcells, e.g., in accordance with a method described herein. In someembodiments, the differentiation factor can induce the differentiationof PDX1-positive pancreatic progenitor cells into NKX6-1-positivepancreatic progenitor cells, e.g., in accordance with a method describedherein. In some embodiments, the differentiation factor can induce thedifferentiation of NKX6-1-positive pancreatic progenitor cells intoinsulin-positive endocrine cells, e.g., in accordance with a methoddescribed herein. In some embodiments, the differentiation factor caninduce the maturation of insulin-positive endocrine cells into SC-βcells, e.g., in accordance with a method described herein.

At least one differentiation factor described herein can be used alone,or in combination with other differentiation actors, to generate SC-βcells according to the methods as disclosed herein. In some embodiments,at least two, at least three, at least four, at least five, at leastsix, at least seven, at least eight, at least nine, or at least tendifferentiation factors described herein are used in the methods ofgenerating SC-β cells.

Transforming Growth Factor-β (TGF-β) Superfamily

Aspects of the disclosure relate to the use of growth factors from thetransforming growth factor-β (TGF-β) superfamily as differentiationfactors. The “TGF-β superfamily” means proteins having structural andfunctional characteristics of known TGFβ family members. The TGFβ familyof proteins can include the TGFβ series of proteins, the Inhibins(including Inhibin A and Inhibin B), the Activins (including Activin A,Activin B, and Activin AB), MIS (Mllerian inhibiting substance), BMP(bone morphogenetic proteins), dpp (decapentaplegic), Vg-1, MNSF(monoclonal nonspecific suppressor factor), and others. Activity of thisfamily of proteins can be based on specific binding to certain receptorson various cell types. Members of this family can share regions ofsequence identity, particularly at the C-terminus, that correlate totheir function. The TGFβ family can include more than one hundreddistinct proteins, all sharing at least one region of amino acidsequence identity. Members of the family that can be used in the methoddisclosed herein can include, but are not limited to, the followingproteins, as identified by their GenBank accession numbers: P07995,P18331, P08476, Q04998, P03970, P43032, P55102, P27092, P42917, P09529,P27093, P04088, Q04999, P17491, P55104, Q9WUK5, P55103, O88959, O08717,P58166, O61643, P35621, P09534, P48970, Q9NR23, P25703, P30884, P12643,P49001, P21274, O46564, O19006, P22004, P20722, Q04906, Q07104, P30886,P18075, P23359, P22003, P34821, P49003, Q90751, P21275, Q06826, P30885,P34820, Q29607, P12644, Q90752, O46576, P27539, P48969, Q26974, P07713,P91706, P91699, P27091, O42222, Q24735, P20863, O18828, P55106, Q9PTQ2,O14793, O08689, O42221, O18830, O18831, O18836, O35312, O42220, P43026,P43027, P43029, O95390, Q9R229, O93449, Q9Z1W4, Q9BDW8, P43028, Q7Z4P5,P50414, P17246, P54831, P04202, P01137, P09533, P18341, O19011, Q9Z1Y6,P07200, Q9Z217, O95393, P55105, P30371, Q9MZE2, Q07258, Q96S42, P97737,AAA97415.1, NP-776788.1, NP-058824.1, EAL24001.1, 1 S4Y, NP-001009856.1,NP-1-032406.1, NP-999193.1, XP-519063.1, AAG17260.1, CAA40806.1,NP-1-001009458.1, AAQ55808.1, AAK40341.1, AAP33019.1, AAK21265.1,AAC59738.1, CAI46003.1, B40905, AAQ55811.1, AAK40342.1, XP-540364.1,P55102, AAQ55810.1, NP-990727.1, CAA51163.1, AAD50448.1, JC4862, PN0504,BAB17600.1, AAH56742.1, BAB17596.1, CAG06183.1, CAG05339.1, BAB17601.1,CAB43091.1, A36192, AAA49162.1, AAT42200.1, NP-789822.1, AAA59451.1,AAA59169.1, XP-541000.1, NP-990537.1, NP-1-002184.1, AAC14187.1,AAP83319.1, AAA59170.1, BAB16973.1, AAM66766.1, WFPGBB, 1201278C,AAH30029.1, CAA49326.1, XP-344131.1, AA-148845.1, XP-1-148966.3, 148235,B41398, AAH77857.1, AAB26863.1, 1706327A, BAA83804.1, NP-571143.1,CAG00858.1, BAB17599.1, BAB17602.1, AAB61468.1, PN0505, PN0506,CAB43092.1, BAB17598.1, BAA22570.1, BAB16972.1, BAC81672.1, BAA12694.1,BAA08494.1, B36192, C36192, BAB16971.1, NP-034695.1, AAA49160.1,CAA62347.1, AAA49161.1, AAD30132.1, CAA58290.1, NP-005529.1,XP-522443.1, AAM27448.1, XP-538247.1, AAD30133. I, AAC36741.1,AAH10404.1, NP-032408.1, AAN03682.1, XP-509161.1, AAC32311.1,NP-651942.2, AAL51005.1, AAC39083.1, AAH85547.1, NP-571023.1,CAF94113.1, EAL29247.1, AAW30007.1, AAH90232.1, A29619, NP-001007905.1,AAH73508.1, AAD02201.1, NP-999793.1, NP-990542.1, AAF19841.1,AAC97488.1, AAC60038.1, NP 989197.1, NP-571434.1, EAL41229.1,AAT07302.1, CAI19472.1, NP-031582.1, AAA40548.1, XP-535880.1,NP-1-037239.1, AAT72007.1, XP-418956.1, CAA41634.1, BAC30864.1,CAA38850.1, CAB81657.2, CAA45018.1, CAA45019.1, BAC28247.1, NP-031581.1,NP-990479.1, NP-999820.1, AAB27335.1, 545355, CAB82007.1, XP-534351.1,NP-058874.1, NP-031579.1, 1REW, AAB96785.1, AAB46367.1, CAA05033.1,BAA89012.1, IES7, AAP20870.1, BAC24087.1, AAG09784.1, BAC06352.1,AAQ89234.1, AAM27000.1, AAH30959.1, CAGO1491.1, NP-571435.1, 1REU,AAC60286.1, BAA24406.1, A36193, AAH55959.1, AAH54647.1, AAH90689.1,CAG09422.1, BAD16743.1, NP-032134.1, XP-532179.1, AAB24876.1,AAH57702.1, AAA82616.1, CAA40222.1, CAB90273.2, XP-342592.1,XP-534896.1, XP-534462.1, 1LXI, XP-417496.1, AAF34179.1, AAL73188.1,CAF96266.1, AAB34226.1, AAB33846.1, AAT12415.1, AA033819.1, AAT72008.1,AAD38402.1, BAB68396.1, CAA45021.1, AAB27337.1, AAP69917.1, AATI2416.1,NP-571396.1, CAA53513.1, AA033820.1, AAA48568.1, BAC02605.1, BAC02604.1,BAC02603.1, BAC02602.1, BAC02601.1, BAC02599.1, BAC02598.1, BAC02597.1,BAC02595.1, BAC02593.1, BAC02592.1, BAC02590.1, AAD28039.1, AAP74560.1,AAB94786.1, NP-001483.2, XP-528195.1, NP-571417.1, NP-001001557. I,AAH43222.1, AAM33143.1, CAG10381.1, BAA31132.1, EAL39680.1, EAA12482.2,P34820, AAP88972.1, AAP74559.1, CAI16418.1, AAD30538.1, XP-345502.1,NP-1-038554.1, CAG04089.1, CAD60936.2, NP-031584.1, B55452, AAC60285.1,BAA06410.1, AAH52846.1, NP-031580.1, NP-1-036959.1, CAA45836.1,CAA45020.1, Q29607, AAB27336.1, XP-547817.1, AAT12414.1, AAM54049.1,AAH78901.1, AA025745.1, NP-570912.1, XP-392194.1, AAD20829.1,AAC97113.1, AAC61694.1, AAH60340.1, AAR97906.1, BAA32227.1, BAB68395.1,BAC02895.1, AAWS 1451.1, AAF82188.1, XP-544189.1, NP-990568.1,BAC80211.1, AAW82620.1, AAF99597.1, NP-571062.1, CAC44179.1, AAB97467.1,AAT99303.1, AAD28038.1, AAH52168.1, NP-001004122.1, CAA72733.1,NP-032133.2, XP-394252.1, XP-224733.2, JH0801, AAP97721.1, NP-989669.1,543296, P43029, A55452, AAH32495.1, XP-542974.1, NP-032135.1,AAK30842.1, AAK27794.1, BAC30847.1, EAA12064.2, AAP97720.1, XP-525704.1,AAT07301.1, BAD07014.1, CAF94356.1, AAR27581.1, AAG13400.1, AAC60127.1,CAF92055.1, XP-540103.1, AA020895.1, CAF97447.1, AAS01764.1, BAD08319.1,CAA10268.1, NP-998140.1, AARO3824.1, AAS48405.1, AAS48403.1, AAK53545.1,AAK84666.1, XP-395420.1, AAK56941.1, AAC47555.1, AAR88255.1, EAL33036.1,AAW47740.1, AAW29442.1, NP-722813.1, AARO8901.1, AAO 15420.2,CAC59700.1, AAL26886.1, AAK71708.1, AAK71707.1, CAC51427.2, AAK67984.1,AAK67983.1, AAK28706.1, P07713, P91706, P91699, CAG02450.1, AAC47552.1,NP-005802.1, XP-343149.1, AW34055.1, XP-538221.1, AAR27580.1,XP-125935.3, AAF21633.1, AAF21630.1, AAD05267.1, Q9Z1 W4, NP-1-031585.2,NP-571094.1, CAD43439.1, CAF99217.1, CAB63584.1, NP-722840.1,CAE46407.1, XP-1-417667.1, BAC53989.1, BAB19659.1, AAM46922.1,AAA81169.1, AAK28707.1, AAL05943.1, AAB17573.1, CAH25443.1, CAG10269.1,BAD16731.1, EAA00276.2, AAT07320.1, AAT07300.1, AAN15037.1, CAH25442.1,AAK08152.2, 2009388A, AAR12161.1, CAGO1961.1, CAB63656.1, CAD67714.1,CAF94162.1, NP-477340.1, EAL24792.1, NP-1-001009428.1, AAB86686.1,AAT40572.1, AAT40571.1, AAT40569.1, NP-033886.1, AAB49985.1, AAG39266.1,Q26974, AAC77461.1, AAC47262.1, BAC05509.1, NP-055297.1, XP-546146.1,XP-525772.1, NP-060525.2, AAH33585.1, AAH69080.1, CAG12751.1,AAH74757.2, NP-034964.1, NP-038639.1, O42221, AAF02773.1, NP-062024.1,AAR18244.1, AAR14343.1, XP-228285.2, AAT40573.1, AAT94456.1, AAL35278.1,AAL35277.1, AAL17640.1, AAC08035.1, AAB86692.1, CAB40844.1, BAC38637.1,BAB16046.1, AAN63522.1, NP-571041.1, AAB04986.2, AAC26791.1, AAB95254.1,BAA11835.1, AAR18246.1, XP-538528.1, BAA31853.1, AAK18000.1,XP-1-420540.1, AAL35276.1, AAQ98602.1, CAE71944.1, AAW50585.1,AAV63982.1, AAW29941.1, AAN87890.1, AAT40568.1, CAD57730.1, AAB81508.1,AAS00534.1, AAC59736.1, BAB79498.1, AAA97392.1, AAP85526.1, NP-999600.2,NP-878293.1, BAC82629.1, CAC60268.1, CAG04919.1, AAN10123.1, CAA07707.1AAK20912.1, AAR88254.1, CAC34629.1, AAL35275.1, AAD46997. I, AAN03842.1,NP-571951.2, CAC50881.1, AAL99367.1, AAL49502.1, AAB71839.1, AAB65415.1,NP-624359.1, NP-990153.1, AAF78069.1, AAK49790.1, NP-919367.2,NP-001192.1, XP-544948.1, AAQ18013.1, AAV38739.1, NP-851298.1,CAA67685.1, AAT67171.1, AAT37502.1, AAD27804.1, AAN76665.1, BAC11909.1,XP-1-421648.1, CAB63704.1, NP-037306.1, A55706, AAF02780.1, CAG09623.1,NP-067589.1, NP-035707.1, AAV30547.1, AAP49817.1, BAC77407.1,AAL87199.1, CAG07172.1, B36193, CAA33024.1, NP-1-001009400.1,AAP36538.1, XP-512687.1, XP-510080.1, AAH05513.1, 1KTZ, AAH14690.1,AAA31526.1.

The growth factor from the TGF-β superfamily in the methods andcompositions provided herein can be naturally obtained or recombinant.In some embodiments, the growth factor from the TGF-β superfamilycomprises Activin A. The term “Activin A” can include fragments andderivatives of Activin A. The sequence of an exemplary Activin A isdisclosed as SEQ ID NO: 1 in U.S. Pub. No. 2009/0155218 (the '218publication). Other non-limiting examples of Activin A are provided inSEQ ID NO: 2-16 of the '218 publication, and non-limiting examples ofnucleic acids encoding Activin A are provided in SEQ ID NO:33-34 of the'218 publication. In some embodiments, the growth factor from the TGF-βsuperfamily can comprise a polypeptide having an amino acid sequence atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or at least 99%, or greateridentical to SEQ ID NO: 1 of the '218 publication.

In some embodiments, the growth factor from the TGF-β superfamilycomprises growth differentiation factor 8 (GDF8). The term “GDF8” caninclude fragments and derivatives of GDF8. The sequences of GDF8polypeptides are available to the skilled artisan. In some embodiments,the growth factor from the TGF-β superfamily comprises a polypeptidehaving an amino acid sequence at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, orat least 99%, or greater identical to the human GDF8 polypeptidesequence (GenBank Accession EAX10880).

In some embodiments, the growth factor from the TGF-β superfamilycomprises a growth factor that is closely related to GDF8, e.g., growthdifferentiation factor 11 (GDF11). In some embodiments, the growthfactor from the TGF-β superfamily comprises a polypeptide having anamino acid sequence at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least99%, or greater identical to the human GDF11 polypeptide sequence(GenBank Accession AAF21630).

In some embodiments, the growth factor from the TGF-β superfamily can bereplaced with an agent mimics the at least one growth factor from theTGF-β superfamily. Exemplary agents that mimic the at least one growthfactor from the TGF-β superfamily, include, without limitation, IDE1 andIDE2.

Bone Morphogenetic Protein (BMP) Signaling Pathway Inhibitors

Aspects of the disclosure relate to the use of BMP signaling pathwayinhibitors as β cell differentiation factors. The BMP signaling familyis a diverse subset of the TGF-β superfamily (Sebald et al. Biol. Chem.385:697-710, 2004). Over twenty known BMP ligands are recognized bythree distinct type II (BMPRII, ActRIIa, and ActRIIb) and at least threetype I (ALK2, ALK3, and ALK6) receptors. Dimeric ligands facilitateassembly of receptor heteromers, allowing the constitutively-active typeII receptor serine/threonine kinases to phosphorylate type I receptorserine/threonine kinases. Activated type I receptors phosphorylateBMP-responsive (BR-) SMAD effectors (SMADs 1, 5, and 8) to facilitatenuclear translocation in complex with SMAD4, a co-SMAD that alsofacilitates TGF signaling. In addition, BMP signals can activateintracellular effectors such as MAPK p38 in a SMAD-independent manner(Nohe et al. Cell Signal 16:291-299, 2004). Soluble BMP antagonists suchas noggin, chordin, gremlin, and follistatin limit BMP signaling byligand sequestration.

In some embodiments, the BMP signaling pathway inhibitor in the methodsand composition provided herein comprises DMH-1, or a derivative,analogue, or variant thereof. In some embodiments, the BMP signalingpathway inhibitor in the methods and composition provided hereincomprises the following compound or a derivative, analogue, or variantof the following compound:

In some embodiments, the BMP signaling pathway inhibitor in the methodsand composition provided herein comprises LDN193189 (also known asLDN193189, 1062368-24-4, LDN-193189, DM 3189, DM-3189, IUPAC Name:4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinolone).In some embodiments, the BMP signaling pathway inhibitor in the methodsand composition provided herein comprises the following compound or aderivative, analogue, or variant of the following compound:

In some cases, DMH-1 can be more selective as compared to LDN193189. Insome embodiments of the present disclosure, DMH-1 can be particularlyuseful for the methods provided herein. In some embodiments, the methodsand compositions provided herein exclude use of LDN193189. In someembodiments, the methods and compositions provided herein exclude use ofLDN193189, or a derivative, analogue, or variant thereof for generatingPDX1-positive pancreatic progenitor cells from primitive gut tube cells.In some embodiments, the methods and compositions provided herein relateto use of DMH-1, or a derivative, analogue, or variant thereof forgenerating PDX1-positive pancreatic progenitor cells from primitive guttube cells.

In some embodiments, the BMP signaling pathway inhibitor in the methodsand composition provided herein comprise an analog or derivative ofLDN193189, e.g., a salt, hydrate, solvent, ester, or prodrug ofLDN193189. In some embodiments, a derivative (e.g., salt) of LDN193189comprises LDN193189 hydrochloride.

In some embodiments, the BMP signaling pathway inhibitor in the methodsand composition provided herein comprises a compound of Formula I fromU.S. Patent Publication No. 2011/0053930.

TGF-β Signaling Pathway Inhibitors

Aspects of the disclosure relate to the use of TGF-β signaling pathwayinhibitors as f3 cell differentiation factors.

In some embodiments, the TGF-β signaling pathway comprises TGF-βreceptor type I kinase (TGF-β RI) signaling. In some embodiments, theTGF-β signaling pathway inhibitor comprises ALK5 inhibitor II (CAS446859-33-2, an ATP-competitive inhibitor of TGF-B RI kinase, also knownas RepSox, IUPAC Name:2-[5-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine. In someembodiments, the TGF-β signaling pathway inhibitor is an analog orderivative of ALK5 inhibitor II.

In some embodiments, the analog or derivative of ALK5 inhibitor II (alsonamed “ALK5i”) is a compound of Formula I as described in U.S. PatentPublication No. 2012/0021519, incorporated by reference herein in itsentirety.

In some embodiments, the TGF-β signaling pathway inhibitor in themethods and compositions provided herein is a TGF-β receptor inhibitordescribed in U.S. Patent Publication No. 2010/0267731. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein comprises an ALK5 inhibitor described inU.S. Patent Publication Nos. 2009/0186076 and 2007/0142376. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is A 83-01. In some embodiments, the TGF-βsignaling pathway inhibitor in the methods and compositions providedherein is not A 83-01. In some embodiments, the compositions and methodsdescribed herein exclude A 83-01. In some embodiments, the TGF-βsignaling pathway inhibitor in the methods and compositions providedherein is SB 431542. In some embodiments, the TGF-β signaling pathwayinhibitor is not SB 431542. In some embodiments, the compositions andmethods described herein exclude SB 431542. In some embodiments, theTGF-β signaling pathway inhibitor in the methods and compositionsprovided herein is D 4476. In some embodiments, the TGF-β signalingpathway inhibitor is not D 4476. In some embodiments, the compositionsand methods described herein exclude D 4476. In some embodiments, theTGF-β signaling pathway inhibitor in the methods and compositionsprovided herein is GW 788388. In some embodiments, the TGF-β signalingpathway inhibitor is not GW 788388. In some embodiments, thecompositions and methods described herein exclude GW 788388. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is LY 364947. In some embodiments, theTGF-β signaling pathway inhibitor is not LY 364947. In some embodiments,the compositions and methods described herein exclude LY 364947. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is LY 580276. In some embodiments, theTGF-β signaling pathway inhibitor is not LY 580276. In some embodiments,the compositions and methods described herein exclude LY 580276. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is SB 525334. In some embodiments, theTGF-β signaling pathway inhibitor is not SB 525334. In some embodiments,the compositions and methods described herein exclude SB 525334. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is SB 505124. In some embodiments, theTGF-β signaling pathway inhibitor is not SB 505124. In some embodiments,the compositions and methods described herein exclude SB 505124. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is SD 208. In some embodiments, the TGF-βsignaling pathway inhibitor is not SD 208. In some embodiments, thecompositions and methods described herein exclude SD 208. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is GW 6604. In some embodiments, the TGF-βsignaling pathway inhibitor is not GW 6604. In some embodiments, thecompositions and methods described herein exclude GW 6604. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is GW 788388. In some embodiments, theTGF-β signaling pathway inhibitor in the methods and compositionsprovided herein is not GW 788388. In some embodiments, the compositionsand methods described herein exclude GW 788388.

From the collection of compounds described above, the following can beobtained from various sources: LY-364947, SB-525334, SD-208, andSB-505124 available from Sigma, P.O. Box 14508, St. Louis, Mo.,63178-9916; 616452 and 616453 available from Calbiochem (EMD Chemicals,Inc.), 480 S. Democrat Road, Gibbstown, N.J., 08027; GW788388 and GW6604available from GlaxoSmithKline, 980 Great West Road, Brentford,Middlesex, TW8 9GS, United Kingdom; LY580276 available from LillyResearch, Indianapolis, Ind. 46285; and SM16 available from Biogen Idec,P.O. Box 14627, 5000 Davis Drive, Research Triangle Park, N.C.,27709-4627.

WNT Signaling Pathway

Aspects of the disclosure relate to the use of activators of the WNTsignaling pathway as β cell differentiation factors.

In some embodiments, the WNT signaling pathway activator in the methodsand compositions provided herein comprises CHIR99021. In someembodiments, the WNT signaling pathway activator in the methods andcompositions provided herein comprises a derivative of CHIR99021, e.g.,a salt of CHIR99021, e.g., trihydrochloride, a hydrochloride salt ofCHIR99021. In some embodiments, the WNT signaling pathway activator inthe methods and compositions provided herein comprises Wnt3a recombinantprotein. In some embodiments, the WNT signaling pathway activator in themethods and compositions provided herein comprises a glycogen synthasekinase 3 (GSK3) inhibitor. Exemplary GSK3 inhibitors include, withoutlimitation, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, FRATide,10Z-Hymenialdisine, Indirubin-3′oxime, kenpaullone, L803, L803-mts,lithium carbonate, NSC 693868, SB 216763, SB 415286, TC-G 24, TCS 2002,TCS 21311, TWS 119, and analogs or derivatives of any of these. Incertain embodiments, the methods, compositions, and kits disclosedherein exclude a WNT signaling pathway activator.

Fibroblast Growth Factor (FGF) Family

Aspects of the disclosure relate to the use of growth factors from theFGF family as β cell differentiation factors.

In some embodiments, the growth factor from the FGF family in themethods and compositions provided herein comprises keratinocyte growthfactor (KGF). The polypeptide sequences of KGF are available to theskilled artisan. In some embodiments, the growth factor from the FGFfamily comprises a polypeptide having an amino acid sequence at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or at least 99%, or greater identicalto the human KGF polypeptide sequence (GenBank Accession AAB21431).

In some embodiments, the growth factor from the FGF family in themethods and composition provided herein comprises FGF2. The polypeptidesequences of FGF2 are available to the skilled artisan. In someembodiments, the growth factor from the FGF family comprises apolypeptide having an amino acid sequence at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 99%, or greater identical to the human FGF2polypeptide sequence (GenBank Accession NP 001997).

In some embodiments, the at least one growth factor from the FGF familyin the methods and composition provided herein comprises FGF8B. Thepolypeptide sequences of FGF8B are available to the skilled artisan. Insome embodiments, the growth factor from the FGF family comprises apolypeptide having an amino acid sequence at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 99%, or greater identical to the human FGF8Bpolypeptide sequence (GenBank Accession AAB40954).

In some embodiments, the at least one growth factor from the FGF familyin the methods and composition provided herein comprises FGF10. Thepolypeptide sequences of FGF10 are available to the skilled artisan. Insome embodiments, the growth factor from the FGF family comprises apolypeptide having an amino acid sequence at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 99%, or greater identical to the human FGF10polypeptide sequence (GenBank Accession CAG46489).

In some embodiments, the at least one growth factor from the FGF familyin the methods and composition provided herein comprises FGF21. Thepolypeptide sequences of FGF21 are available to the skilled artisan. Insome embodiments, the growth factor from the FGF family comprises apolypeptide having an amino acid sequence at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 99%, or greater identical to the human FGF21polypeptide sequence (GenBank Accession AAQ89444.1).

Sonic Hedgehog (SHH) Signaling Pathway

Aspects of the disclosure relate to the use of SHH signaling pathwayinhibitors as β cell differentiation factors.

In some embodiments, the SHH signaling pathway inhibitor in the methodsand composition provided herein comprises Sant1. In some embodiments,the SHH signaling pathway inhibitor in the methods and compositionprovided herein comprises SANT2. In some embodiments, the SHH signalingpathway inhibitor in the methods and composition provided hereincomprises SANT3. In some embodiments, the SHH signaling pathwayinhibitor in the methods and composition provided herein comprisesSANT4. In some embodiments, the SHH signaling pathway inhibitorcomprises Cur61414. In some embodiments, the SHH signaling pathwayinhibitor in the methods and composition provided herein comprisesforskolin. In some embodiments, the SHH signaling pathway inhibitor inthe methods and composition provided herein comprises tomatidine. Insome embodiments, the SHH signaling pathway inhibitor in the methods andcomposition provided herein comprises AY9944. In some embodiments, theSHH signaling pathway inhibitor in the methods and composition providedherein comprises triparanol. In some embodiments, the SHH signalingpathway inhibitor in the methods and composition provided hereincomprises compound A or compound B (as disclosed in U.S. Pub. No.2004/0060568). In some embodiments, the SHH signaling pathway inhibitorin the methods and composition provided herein comprises a steroidalalkaloid that antagonizes hedgehog signaling (e.g., cyclopamine or aderivative thereof) as disclosed in U.S. Pub. No. 2006/0276391. Incertain embodiments, the methods, compositions, and kits disclosedherein exclude a SHH signaling pathway inhibitor.

Rho Kinase (ROCK) Signaling Pathway

Aspects of the disclosure relate to the use of ROCK signaling pathwayinhibitors (ROCK inhibitors) as β cell differentiation factors.

In some embodiments, the ROCK inhibitor in the methods and compositionprovided herein comprises Y-27632 or Thiazovivin. In some embodiments,the ROCK inhibitor in the methods and composition provided hereincomprises Thiazovivin. In some embodiments, the ROCK inhibitor in themethods and composition provided herein comprises Y-27632. In somecases, the ROCK inhibitor in the methods and composition provided hereincomprises the following compound or a derivative thereof:

In some cases, the ROCK inhibitor in the methods and compositionprovided herein comprises the following compound or a derivativethereof:

Non-limiting examples of ROCK inhibitor that can be used in the methodsand compositions provided herein include Thiazovivin, Y-27632,Fasudil/HA1077, H-1152, Ripasudil, Y39983, Wf-536, SLx-2119,Azabenzimidazole-aminofurazans, DE-104, Olefins, Isoquinolines,Indazoles, and pyridinealkene derivatives, ROKα inhibitor, XD-4000,HMN-1152, 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides,Rhostatin, BA-210, BA-207, BA-215, BA-285, BA-1037, Ki-23095, VAS-012,and quinazoline.

Retinoic Acid Signaling Pathway

Aspects of the disclosure relate to the use of modulators of retinoicacid signaling as β cell differentiation factors.

In some embodiments, the modulator of retinoic acid signaling in themethods and composition provided herein comprises an activator ofretinoic acid signaling. In some embodiments, the RA signaling pathwayactivator in the methods and composition provided herein comprisesretinoic acid. In some embodiments, the RA signaling pathway activatorin the methods and composition provided herein comprises a retinoic acidreceptor agonist. Exemplary retinoic acid receptor agonists in themethods and composition provided herein include, without limitation, CD1530, AM 580, TTNPB, CD 437, Ch 55, BMS 961, AC 261066, AC 55649, AM 80,BMS 753, tazarotene, adapalene, and CD 2314.

In some embodiments, the modulator of retinoic acid signaling in themethods and composition provided herein comprises an inhibitor ofretinoic acid signaling. In some embodiments, the retinoic acidsignaling pathway inhibitor comprises DEAB (IUPAC Name:2-[2-(diethylamino)ethoxy]-3-prop-2-enylbenzaldehyde). In someembodiments, the retinoic acid signaling pathway inhibitor comprises ananalog or derivative of DEAB.

In some embodiments, the retinoic acid signaling pathway inhibitor inthe methods and composition provided herein comprises a retinoic acidreceptor antagonist. In some embodiments, the retinoic acid receptorantagonist in the methods and composition provided herein comprises(E)-4-[2-(5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethenyl]benzoicacid,(E)-4-[[(5,6-dihydro-5,5-dimethyl-8-phenylethynyl)-2-naphthalenyl]ethenyl]benzoicacid,(E)-4-[2-[5,6-dihydro-5,5-dimethyl-8-(2-naphthalenyl)-2-naphthalenyl]ethenyl]-benzoicacid, and(E)-4-[2-[5,6-dihydro-5,5-dimethyl-8-(4-methoxyphenyl)-2-naphthalenyl]ethenyl]benzoicacid. In some embodiments, the retinoic acid receptor antagonistcomprises BMS 195614 (CAS#253310-42-8), ER 50891 (CAS#187400-85-7), BMS493 (CAS#170355-78-9), CD 2665 (CAS#170355-78-9), LE 135(CAS#155877-83-1), BMS 453 (CAS #166977-43-1), or MM 11253(CAS#345952-44-5).

In certain embodiments, the methods, compositions, and kits disclosedherein exclude a modulator of retinoic acid signaling. In certainembodiments, the methods, compositions, and kits disclosed hereinexclude a retinoic acid signaling pathway activator. In certainembodiments, the methods, compositions, and kits disclosed hereinexclude a retinoic acid signaling pathway inhibitor.

Protein Kinase C

Aspects of the disclosure relate to the use of protein kinase Cactivators as β cell differentiation factors. Protein kinase C is one ofthe largest families of protein kinase enzymes and is composed of avariety of isoforms. Conventional isoforms include a, βI, βII, γ; novelisoforms include δ, ε, η, Θ; and atypical isoforms include ξ, and 1/λ.PKC enzymes are primarily cytosolic but translocate to the membrane whenactivated. In the cytoplasm, PKC is phosphorylated by other kinases orautophosphorylated. In order to be activated, some PKC isoforms (e.g.,PKC-ε) require a molecule to bind to the diacylglycerol (“DAG”) bindingsite or the phosphatidylserine (“PS”) binding site. Others are able tobe activated without any secondary binding messengers at all. PKCactivators that bind to the DAG site include, but are not limited to,bryostatin, picologues, phorbol esters, aplysiatoxin, and gnidimacrin.PKC activators that bind to the PS site include, but are not limited to,polyunsaturated fatty acids and their derivatives. It is contemplatedthat any protein kinase C activator that is capable, either alone or incombination with one or more other β cell differentiation factors, ofinducing the differentiation of at least one insulin-producing,endocrine cell or precursor thereof into a SC-β cell can be used in themethods, compositions, and kits described herein.

In some embodiments, any of the PKC activators disclosed herein is a PKCactivator capable of binding to a DAG binding site on a PKC. In someembodiments, the PKC activator is capable of binding to a Cl domain of aPKC. In some embodiments, the PKC activator is a benzolactam-derivative.In some embodiments, the benzolactam-derivative is((2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam),which may be referred to herein as TPPB or TPB. In some embodiments,contacting a population of cells with a benzolactam-derivative PKCactivator (e.g., TPPB) increases cell yield as compared to a populationof cells not treated with the benzolactam-derivative PKC activator. Insome embodiments, the PKC activator is a phorbol ester. In someembodiments, the phorbol ester is Phorbol 12,13-dibutyrate, which may bereferred to herein as PDBU or PdbU. In some embodiments, contacting apopulation of cells with a benzolactam-derivative PKC activator (e.g.,TPPB) increases cell yield as compared to a population of cells treatedwith a phorbol ester PKC activator (e.g., PdbU). In some embodiments,the PKC activator in the methods and composition provided hereincomprises PdbU. In some embodiments, the PKC activator in the methodsand composition provided herein comprises TPB. In some embodiments, thePKC activator in the methods and composition provided herein comprisescyclopropanated polyunsaturated fatty acids, cyclopropanatedmonounsaturated fatty acids, cyclopropanated polyunsaturated fattyalcohols, cyclopropanated monounsaturated fatty alcohols,cyclopropanated polyunsaturated fatty acid esters, cyclopropanatedmonounsaturated fatty acid esters, cyclopropanated polyunsaturated fattyacid sulfates, cyclopropanated monounsaturated fatty acid sulfates,cyclopropanated polyunsaturated fatty acid phosphates, cyclopropanatedmonounsaturated fatty acid phosphates, macrocyclic lactones, DAGderivatives, isoprenoids, octylindolactam V, gnidimacrin, iripallidal,ingenol, napthalenesulfonamides, diacylglycerol kinase inhibitors,fibroblast growth factor 18 (FGF-18), insulin growth factor, hormones,and growth factor activators, as described in WIPO Pub. No.WO/2013/071282. In some embodiments, the bryostain comprisesbryostatin-1, bryostatin-2, bryostatin-3, bryostatin-4, bryostatin-5,bryostatin-6, bryostatin-7, bryostatin-8, bryostatin-9, bryostatin-10,bryostatin-11, bryostatin-12, bryostatin-13, bryostatin-14,bryostatin-15, bryostatin-16, bryostatin-17, or bryostatin-18. Incertain embodiments, the methods, compositions, and kits disclosedherein exclude a protein kinase C activator.

γ-Secretase Inhibitors

Aspects of the disclosure relate to the use of γ-secretase inhibitors asβ cell differentiation factors.

In some embodiments, the γ-secretase inhibitor in the methods andcomposition provided herein comprises XXI. In some embodiments, theγ-secretase inhibitor in the methods and composition provided hereincomprises DAPT. Additional exemplary γ-secretase inhibitors in themethods and composition provided herein include, without limitation, theγ-secretase inhibitors described in U.S. Pat. Nos. 7,049,296, 8,481,499,8,501,813, and WIPO Pub. No. WO/2013/052700. In certain embodiments, themethods, compositions, and kits disclosed herein exclude a γ-secretaseinhibitor.

Thyroid Hormone Signaling Pathway Activators

Aspects of the disclosure relate to the use of thyroid hormone signalingpathway activators as β cell differentiation factors.

In some embodiments, the thyroid hormone signaling pathway activator inthe methods and composition provided herein comprises triiodothyronine(T3). In some embodiments, the thyroid hormone signaling pathwayactivator in the methods and composition provided herein comprises GC-1.In some embodiments, the thyroid hormone signaling pathway activator inthe methods and composition provided herein comprises an analog orderivative of T3 or GC-1. Exemplary analogs of T3 in the methods andcomposition provided herein include, but are not limited to, selectiveand non-selective thyromimetics, TRβ selective agonist-GC-1,GC-24,4-Hydroxy-PCB 106, MB07811, MB07344,3,5-diiodothyropropionic acid(DITPA); the selective TR-β agonist GC-1; 3-Iodothyronamine (T(1)AM) and3,3′,5-triiodothyroacetic acid (Triac) (bioactive metabolites of thehormone thyroxine (T(4)); KB-2115 and KB-141; thyronamines; SKF L-94901;DIBIT; 3′-AC-T2; tetraiodothyroacetic acid (Tetrac) andtriiodothyroacetic acid (Triac) (via oxidative deamination anddecarboxylation of thyroxine [T4] and triiodothyronine [T3] alaninechain), 3,3′,5′-triiodothyronine (rT3) (via T4 and T3 deiodination),3,3′-diiodothyronine (3,3′-T2) and 3,5-diiodothyronine (T2) (via T4, T3,and rT3 deiodination), and 3-iodothyronamine (T1AM) and thyronamine(T0AM) (via T4 and T3 deiodination and amino acid decarboxylation), aswell as for TH structural analogs, such as 3,5,3′-triiodothyropropionicacid (Triprop), 3,5-dibromo-3-pyridazinone-1-thyronine (L-940901),N-[3,5-dimethyl-4-(4′-hydroxy-3′-isopropylphenoxy)-phenyl]-oxamic acid(CGS 23425),3,5-dimethyl-4-[(4′-hydroxy-3′-isopropylbenzyl)-phenoxy]acetic acid(GC-1), 3,5-dichloro-4-[(4-hydroxy-3-isopropylphenoxy)phenyl]acetic acid(KB-141), and 3,5-diiodothyropropionic acid (DITPA).

In some embodiments, the thyroid hormone signaling pathway activator inthe methods and composition provided herein comprises a prodrug orprohormone of T3, such as T4 thyroid hormone (e.g., thyroxine orL-3,5,3′,5′-tetraiodothyronine).

In some embodiments, the thyroid hormone signaling pathway activator inthe methods and composition provided herein is an iodothyroninecomposition described in U.S. Pat. No. 7,163,918.

Epidermal Growth Factor (EGF) Family

Aspects of the disclosure relate to the use of growth factors from theEGF family as β cell differentiation factors.

In some embodiments, the at least one growth factor from the EGF familyin the methods and composition provided herein comprises betacellulin.In some embodiments, at least one growth factor from the EGF family inthe methods and composition provided herein comprises EGF. Epidermalgrowth factor (EGF) is a 53 amino acid cytokine which is proteolyticallycleaved from a large integral membrane protein precursor. In someembodiments, the growth factor from the EGF family in the methods andcomposition provided herein comprises a variant EGF polypeptide, forexample an isolated epidermal growth factor polypeptide having at least90% amino acid identity to the human wild-type EGF polypeptide sequence,as disclosed in U.S. Pat. No. 7,084,246. In some embodiments, the growthfactor from the EGF family in the methods and composition providedherein comprises an engineered EGF mutant that binds to and agonizes theEGF receptor, as is disclosed in U.S. Pat. No. 8,247,531. In someembodiments, the at least one growth factor from the EGF family in themethods and composition provided herein is replaced with an agent thatactivates a signaling pathway in the EGF family. In some embodiments,the growth factor from the EGF family in the methods and compositionprovided herein comprises a compound that mimics EGF. In certainembodiments, the methods, compositions, and kits disclosed hereinexclude a growth factor from the EGF family.

Epigenetic Modifying Compounds

Aspects of the disclosure relate to the use of epigenetic modifyingcompound as β cell differentiation factors.

The term “epigenetic modifying compound” can refer to a chemicalcompound that can make epigenetic changes genes, i.e., change geneexpression(s) without changing DNA sequences. Epigenetic changes canhelp determine whether genes are turned on or off and can influence theproduction of proteins in certain cells, e.g., beta-cells. Epigeneticmodifications, such as DNA methylation and histone modification, canalter DNA accessibility and chromatin structure, thereby regulatingpatterns of gene expression. These processes can be crucial to normaldevelopment and differentiation of distinct cell lineages in the adultorganism. They can be modified by exogenous influences, and, as such,can contribute to or be the result of environmental alterations ofphenotype or pathophenotype. Importantly, epigenetic modification canhave a crucial role in the regulation of pluripotency genes, whichbecome inactivated during differentiation. Non-limiting exemplaryepigenetic modifying compound include a DNA methylation inhibitor, ahistone acetyltransferase inhibitor, a histone deacetylase inhibitor, ahistone methyltransferase inhibitor, a bromodomain inhibitor, or anycombination thereof.

In an embodiment, the histone methyltransferase inhibitor is aninhibitor of enhancer of zeste homolog 2 (EZH2). EZH2 is ahistone-lysine N-methyltransferase enzyme. Non-limiting examples of anEZH2 inhibitor that can be used in the methods provided herein include3-deazaneplanocin A (DZNep), EPZ6438, EPZ005687 (an S-adenosylmethionine(SAM) competitive inhibitor), EI1, GSK126, and UNC1999. DZNep caninhibit the hydrolysis of S-adenosyl-L-homocysteine (SAH), which is aproduct-based inhibitor of all protein methyltransferases, leading toincreased cellular concentrations of SAH which in turn inhibits EZH2.DZNep may not be specific to EZH2 and can also inhibit other DNAmethyltransferases. GSK126 is a SAM-competitive EZH2 inhibitor that has150-fold selectivity over EZH1. UNC1999 is an analogue of GSK126, and itis less selective than its counterpart GSK126.

In an embodiment, the histone methyltransferase inhibitor is DZNep. Inan embodiment, the HDAC inhibitor is a class I HDAC inhibitor, a classII HDAC inhibitor, or a combination thereof. In an embodiment, the HDACinhibitor is KD5170 (mercaptoketone-based HDAC inhibitor), MC1568 (classIIa HDAC inhibitor), TMP195 (class IIa HDAC inhibitor), or anycombination thereof. In some embodiments, HDAC inhibitor is vorinostat,romidepsin (Istodax), chidamide, panobinostat (farydak), belinostat(PXD101), panobinostat (LBH589), valproic acid, mocetinostat (MGCD0103),abexinostat (PCI-24781), entinostat (MS-275), SB939, resminostat(4SC-201), givinostat (ITF2357), quisinostat (JNJ-26481585), HBI-8000,(a benzamide HDI), kevetrin, CUDC-101, AR-42, CHR-2845, CHR-3996,4SC-202, CG200745, ACY-1215, ME-344, sulforaphane, or any variantthereof.

Protein Kinase Inhibitors

Aspects of the disclosure relate to the use of protein kinase inhibitorsas β cell differentiation factors.

In some embodiments, the protein kinase inhibitor in the methods andcomposition provided herein comprises staurosporine. In someembodiments, the protein kinase inhibitor in the methods and compositionprovided herein comprises an analog of staurosporine. Exemplary analogsof staurosporine in the methods and composition provided herein include,without limitation, Ro-31-8220, a bisindolylmaleimide (B is) compound,10′-{5″-[(methoxycarbonyl)amino]-2″-methyl}-phenylaminocarbonylstaurosporine,a staralog (see, e.g., Lopez et al., “Staurosporine-derived inhibitorsbroaden the scope of analog-sensitive kinase technology”, J. Am. Chem.Soc. 2013; 135(48):18153-18159), and, cgp41251.

In some embodiments, the protein kinase inhibitor in the methods andcomposition provided herein is an inhibitor of PKCβ. In someembodiments, the protein kinase inhibitor in the methods and compositionprovided herein is an inhibitor of PKCβ with the following structure ora derivative, analogue or variant of the compound as follows:

In some embodiments, the inhibitor of PKCβ is a GSK-2 compound with thefollowing structure or a derivative, analogue or variant of the compoundas follows:

In some embodiments, the inhibitor of PKC in the methods and compositionprovided herein is a bisindolylmaleimide. Exemplary bisindolylmaleimidesinclude, without limitation, bisindolylmaleimide I, bisindolylmaleimideII, bisindolylmaleimide Ill, hydrochloride, or a derivative, analogue orvariant thereof.

In some embodiments, the PKC inhibitor in the methods and compositionprovided herein is a pseudohypericin, or a derivative, analogue, orvariant thereof. In some embodiments, the PKC inhibitor in the methodsand composition provided herein is indorublin-3-monoximc, 5-Iodo or aderivative, analogue or variant thereof. In certain embodiments, themethods, compositions, and kits disclosed herein exclude a proteinkinase inhibitor.

Pharmaceutical Compositions

The present disclosure relates to a therapeutic composition containingcells produced by any of the foregoing methods or containing any of theforegoing cell populations. The therapeutic compositions can furthercomprise a physiologically compatible solution including, for example,artificial cerebrospinal fluid or phosphate-buffered saline. Thetherapeutic composition can be used to treat, prevent, or stabilizediabetes. For example, somatic cells or stem cells can be obtained froman individual in need of treatment or from a healthy individual andreprogrammed to stem cell derived beta cells by the method of thepresent disclosure. In one embodiment of the present disclosure the stemcell derived beta cells are sorted and enriched and introduced into theindividual to treat the condition. In another embodiment the stem cellsare cultured under conditions suitable for differentiation into betacells prior to introduction into the individual, and can be used toreplace or assist the normal function of diseased or damaged tissue. Thegreat advantage of the present disclosure is that it provides anessentially limitless supply of patient specific human beta cells orcompatible stem cell derived beta cells from healthy individuals withthe same HLA type suitable for transplantation. The use of autologousand/or compatible cells in cell therapy offers a major advantage overthe use of non-autologous cells, which are likely to be subject toimmunological rejection. In contrast, autologous cells are unlikely toelicit significant immunological responses.

In some cases, the present disclosure provides pharmaceuticalcompositions that can utilize non-native pancreatic β cell (beta cells)populations and cell components and products in various methods fortreatment of a disease (e.g., diabetes). Certain cases encompasspharmaceutical compositions comprising live cells (e.g., non-nativepancreatic β cells alone or admixed with other cell types). Other casesencompass pharmaceutical compositions comprising non-native pancreatic βcell components (e.g., cell lysates, soluble cell fractions, conditionedmedium, ECM, or components of any of the foregoing) or products (e.g.,trophic and other biological factors produced by non-native pancreatic βcells or through genetic modification, conditioned medium fromnon-native pancreatic β cell culture). In either case, thepharmaceutical composition may further comprise other active agents,such as anti-inflammatory agents, exogenous small molecule agonists,exogenous small molecule antagonists, anti-apoptotic agents,antioxidants, and/or growth factors known to a person having skill inthe art.

In some embodiments, any of the cells disclosed herein comprise agenomic disruption in at least one gene sequence, wherein saiddisruption reduces or eliminates expression of a protein encoded by saidgene sequence. In some embodiments, said cells comprise a genomicdisruption in at least one gene sequence, wherein said disruptionreduces or eliminates expression of a protein encoded by said genesequence. In some embodiments, said cells comprise a genomic disruptionin at least one gene sequence, wherein said disruption reduces oreliminates expression of a protein encoded by said gene sequence. Insome embodiments, any of the cells disclosed herein (e.g., any of theSC-derived beta cells or cells in any of the clusters disclosed herein)comprise a genomic disruption in at least one gene sequence, whereinsaid disruption reduces or eliminates expression of a protein encoded bysaid gene sequence. In some embodiments, said at least one gene sequenceencodes an MHC-Class I gene. In some embodiments, said MHC-Class I geneencodes beta-2 microglobulin (B2M), HLA-A, HLA-B, or HLA-C. In someembodiments, said at least one gene sequence encodes CIITA. In someembodiments, said cells comprise a genomic disruption in a naturalkiller cell activating ligand gene. In some embodiments, said naturalkiller cell activating ligand gene encodes intercellular adhesionmolecule 1 (ICAM1), CD58, CD155, carcinoembryonic antigen-related celladhesion molecule 1 (CEACAM1), cell adhesion molecule 1 (CADM1),MHC-Class I polypeptide-related sequence A (MICA), or MHC-Class Ipolypeptide-related sequence B (MICB). In some embodiments, the genomicdisruption is induced by use of a gene editing system, e.g., CRISPR Castechnology.

Pharmaceutical compositions of the present disclosure can comprisenon-native pancreatic β cell, or components or products thereof,formulated with a pharmaceutically acceptable carrier (e.g. a medium oran excipient). The term pharmaceutically acceptable carrier (or medium),which may be used interchangeably with the term biologically compatiblecarrier or medium, can refer to reagents, cells, compounds, materials,compositions, and/or dosage forms that are not only compatible with thecells and other agents to be administered therapeutically, but also aresuitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or othercomplication. Suitable pharmaceutically acceptable carriers can includewater, salt solution (such as Ringer's solution), alcohols, oils,gelatins, and carbohydrates, such as lactose, amylose, or starch, fattyacid esters, hydroxymethylcellulose, and polyvinyl pyrolidine. Suchpreparations can be sterilized, and if desired, mixed with auxiliaryagents such as lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, andcoloring. Pharmaceutical compositions comprising cellular components orproducts, but not live cells, can be formulated as liquids.Pharmaceutical compositions comprising living non-native pancreatic βcells can be formulated as liquids, semisolids (e.g., gels, gelcapsules, or liposomes) or solids (e.g., matrices, scaffolds and thelike).

As used here, the term “pharmaceutically acceptable” can refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used here, the term “pharmaceutically-acceptable carrier” can referto a pharmaceutically-acceptable material, composition or vehicle, suchas a liquid or solid filler, diluent, excipient, manufacturing aid(e.g., lubricant, talc magnesium, calcium or zinc stearate, or stericacid), or solvent encapsulating material, involved in carrying ortransporting the subject compound from one organ, or portion of thebody, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient. Some examples ofmaterials which can serve as pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, methylcellulose,ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, suchas magnesium stearate, sodium lauryl sulfate and talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient,” “carrier,” “pharmaceutically acceptablecarrier” or the like are used interchangeably herein.

The phrase “therapeutically-effective amount” as used herein in respectto a population of cells means that amount of relevant cells in apopulation of cells, e.g., SC-β cells or mature pancreatic β cells, orcomposition comprising SC-β cells of the present disclosure which iseffective for producing some desired therapeutic effect in at least asub-population of cells in an animal at a reasonable benefit/risk ratioapplicable to any medical treatment. For example, an amount of apopulation of SC-β cells administered to a subject that is sufficient toproduce a statistically significant, measurable change in at least onesymptom of Type 1, Type 1.5 or Type 2 diabetes, such as glycosylatedhemoglobin level, fasting blood glucose level, hypoinsulinemia, etc.Determination of a therapeutically effective amount is well within thecapability of those skilled in the art. Generally, a therapeuticallyeffective amount can vary with the subject's history, age, condition,sex, as well as the severity and type of the medical condition in thesubject, and administration of other pharmaceutically active agents.

In some instances, pharmaceutical compositions of the stem cell derivedbeta cells are formulated in a conventional manner using one or morephysiologically acceptable carriers including excipients and auxiliarieswhich facilitate processing of the active compounds into preparationswhich can be used pharmaceutically. Proper formulation is dependent uponthe route of administration chosen. A summary of pharmaceuticalcompositions described herein is found, for example, in Remington: TheScience and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: MackPublishing Company, 1995); Hoover, John E., Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1975; Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York,N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems,Seventh Ed. (Lippincott Williams & Wilkins1999).

Pharmaceutical compositions are optionally manufactured in aconventional manner, such as, by way of example only, by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or compression processes.

In certain embodiments, compositions may also include one or more pHadjusting agents or buffering agents, including acids such as acetic,boric, citric, lactic, phosphoric and hydrochloric acids; bases such assodium hydroxide, sodium phosphate, sodium borate, sodium citrate,sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; andbuffers such as citrate/dextrose, sodium bicarbonate and ammoniumchloride. Such acids, bases and buffers are included in an amountrequired to maintain pH of the composition in an acceptable range.

In other embodiments, compositions can also include one or more salts inan amount required to bring osmolality of the composition into anacceptable range. Such salts include those having sodium, potassium orammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

The pharmaceutical compositions described herein are administered by anysuitable administration route, including but not limited to, oral,parenteral (e.g., intravenous, subcutaneous, intramuscular,intracerebral, intracerebroventricular, intra-articular,intraperitoneal, or intracranial), intranasal, buccal, sublingual, orrectal administration routes. In some instances, the pharmaceuticalcomposition is formulated for parenteral (e.g., intravenous,subcutaneous, intramuscular, intracerebral, intracerebroventricular,intra-articular, intraperitoneal, or intracranial) administration.

The pharmaceutical compositions described herein are formulated into anysuitable dosage form, including but not limited to, aqueous oraldispersions, liquids, gels, syrups, elixirs, slurries, suspensions andthe like, for oral ingestion by an individual to be treated, solid oraldosage forms, aerosols, controlled release formulations, fast meltformulations, effervescent formulations, lyophilized formulations,tablets, powders, pills, dragees, capsules, delayed releaseformulations, extended release formulations, pulsatile releaseformulations, multiparticulate formulations, and mixed immediate releaseand controlled release formulations. In some embodiments, thepharmaceutical compositions are formulated into capsules. In someembodiments, the pharmaceutical compositions are formulated intosolutions (for example, for IV administration). In some cases, thepharmaceutical composition is formulated as an infusion. In some cases,the pharmaceutical composition is formulated as an injection.

The pharmaceutical solid dosage forms described herein optionallyinclude a compound described herein and one or more pharmaceuticallyacceptable additives such as a compatible carrier, binder, fillingagent, suspending agent, flavoring agent, sweetening agent,disintegrating agent, dispersing agent, surfactant, lubricant, colorant,diluent, solubilizer, moistening agent, plasticizer, stabilizer,penetration enhancer, wetting agent, anti-foaming agent, antioxidant,preservative, or one or more combination thereof.

In still other aspects, using standard coating procedures, such as thosedescribed in Remington's Pharmaceutical Sciences, 20th Edition (2000), afilm coating is provided around the compositions. In some embodiments,the compositions are formulated into particles (for example foradministration by capsule) and some or all of the particles are coated.In some embodiments, the compositions are formulated into particles (forexample for administration by capsule) and some or all of the particlesare microencapsulated. In some embodiments, the compositions areformulated into particles (for example for administration by capsule)and some or all of the particles are not microencapsulated and areuncoated.

In certain embodiments, compositions provided herein may also includeone or more preservatives to inhibit microbial activity. Suitablepreservatives include mercury-containing substances such as merfen andthiomersal; stabilized chlorine dioxide; and quaternary ammoniumcompounds such as benzalkonium chloride, cetyltrimethylammonium bromideand cetylpyridinium chloride.

In some embodiments, a composition of the present disclosure cancomprise the stem cell derived beta cells, in an amount that iseffective to treat or prevent e.g., diabetes. A pharmaceuticalcomposition can comprise the stem cell derived beta cells as describedherein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions can comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

Pharmaceutical compositions can comprise auxiliary components as wouldbe familiar to a person having skill in the art. For example, they cancontain antioxidants in ranges that vary depending on the kind ofantioxidant used. Reasonable ranges for commonly used antioxidants areabout 0.01% to about 0.15% weight by volume of EDTA, about 0.01% toabout 2.0% weight volume of sodium sulfite, and about 0.01% to about2.0% weight by volume of sodium metabisulfite. One skilled in the artmay use a concentration of about 0.1% weight by volume for each of theabove. Other representative compounds include mercaptopropionyl glycine,N-acetyl cysteine, P-mercaptoethylamine, glutathione and similarspecies, although other anti-oxidant agents suitable for renaladministration, e.g. ascorbic acid and its salts or sulfite or sodiummetabisulfite may also be employed.

A buffering agent can be used to maintain the pH of formulations in therange of about 4.0 to about 8.0; soas to minimize irritation in thetarget tissue. For direct intraperitoneal injection, formulations shouldbe at pH 7.2 to 7.5, preferably at pH 7.35-7.45. The compositions mayalso include tonicity agents suitable for administration to the kidney.Among those suitable is sodium chloride to make formulationsapproximately isotonic with blood.

In certain cases, pharmaceutical compositions are formulated withviscosity enhancing agents. Exemplary agents are hydroxyethylcellulose,hydroxypropylcellulose, methylcellulose, and polyvinylpyrrolidone. Thepharmaceutical compositions may have cosolvents added if needed.Suitable cosolvents may include glycerin, polyethylene glycol (PEG),polysorbate, propylene glycol, and polyvinyl alcohol. Preservatives mayalso be included, e.g., benzalkonium chloride, benzethonium chloride,chlorobutanol, phenylmercuric acetate or nitrate, thimerosal, or methylor propylparabens.

Pharmaceutical compositions comprising cells, cell components or cellproducts may be delivered to the kidney of a patient in one or more ofseveral methods of delivery known in the art. In some cases, thecompositions are delivered to the kidney (e.g., on the renal capsuleand/or underneath the renal capsule). In another embodiment, thecompositions may be delivered to various locations within the kidney viaperiodic intraperitoneal or intrarenal injection. Alternatively, thecompositions may be applied in other dosage forms known to those skilledin the art, such as pre-formed or in situ-formed gels or liposomes.

Pharmaceutical compositions comprising live cells in a semi-solid orsolid carrier are may be formulated for surgical implantation on orbeneath the renal capsule. It should be appreciated that liquidcompositions also may be administered by surgical procedures. Inparticular cases, semi-solid or solid pharmaceutical compositions maycomprise semi-permeable gels, lattices, cellular scaffolds and the like,which may be non-biodegradable or biodegradable. For example, in certaincases, it may be desirable or appropriate to sequester the exogenouscells from their surroundings, yet enable the cells to secrete anddeliver biological molecules (e.g., insulin) to surrounding cells or theblood stream. In these cases, cells may be formulated as autonomousimplants comprising living non-native pancreatic β cells or cellpopulation comprising non-native pancreatic β cell surrounded by anon-degradable, selectively permeable barrier that physically separatesthe transplanted cells from host tissue. Such implants are sometimesreferred to as “immunoprotective,” as they have the capacity to preventimmune cells and macromolecules from killing the transplanted cells inthe absence of pharmacologically induced immunosuppression.

In other cases, various degradable gels and networks can be used for thepharmaceutical compositions of the present disclosure. For example,degradable materials particularly suitable for sustained releaseformulations include biocompatible polymers, such as poly(lactic acid),poly (lactic-co-glycolic acid), methylcellulose, hyaluronic acid,collagen, and the like.

In other cases, it may be desirable or appropriate to deliver the cellson or in a biodegradable, preferably bioresorbable or bioabsorbable,scaffold or matrix. These typically three-dimensional biomaterialscontain the living cells attached to the scaffold, dispersed within thescaffold, or incorporated in an extracellular matrix entrapped in thescaffold. Once implanted into the target region of the body, theseimplants become integrated with the host tissue, wherein thetransplanted cells gradually become established.

Examples of scaffold or matrix (sometimes referred to collectively as“framework”) material that may be used in the present disclosure includenonwoven mats, porous foams, or self-assembling peptides. Nonwoven mats,for example, may be formed using fibers comprising a syntheticabsorbable copolymer of glycolic and lactic acids (PGA/PLA), foams,and/or poly(epsilon-caprolactone)/poly(glycolic acid) (PCL/PGA)copolymer.

In another embodiment, the framework is a felt, which can be composed ofa multifilament yarn made from a bioabsorbable material, e.g., PGA, PLA,PCL copolymers or blends, or hyaluronic acid. The yarn is made into afelt using standard textile processing techniques consisting ofcrimping, cutting, carding and needling. In another embodiment, cellsare seeded onto foam scaffolds that may be composite structures. In manyof the abovementioned cases, the framework may be molded into a usefulshape. Furthermore, it will be appreciated that non-native pancreatic βcells may be cultured on pre-formed, non-degradable surgical orimplantable devices.

The matrix, scaffold or device may be treated prior to inoculation ofcells in order to enhance cell attachment. For example, prior toinoculation, nylon matrices can be treated with 0.1 molar acetic acidand incubated in polylysine, PBS, and/or collagen to coat the nylon.Polystyrene can be similarly treated using sulfuric acid. The externalsurfaces of a framework may also be modified to improve the attachmentor growth of cells and differentiation of tissue, such as by plasmacoating the framework or addition of one or more proteins (e.g.,collagens, elastic fibers, reticular fibers), glycoproteins,glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate,chondroitin-6-sulfate, dermatan sulfate, keratin sulfate), α cellularmatrix, and/or other materials such as, but not limited to, gelatin,alginates, agar, agarose, and plant gums, among others.

In one aspect, the present disclosure provided devices comprising acellcluster comprising at least one pancreatic β cell. A device providedherein can be configured to produce and release insulin when implantedinto a subject. A device can comprise a cell cluster comprising at leastone pancreatic β cell, e.g., a non-native pancreatic β cell. A cellcluster in the device can exhibit in vitro GSIS. A device can furthercomprise a semipermeable membrane. The semipermeable membrane can beconfigured to retain the cell cluster in the device and permit passageof insulin secreted by the cell cluster. In some cases of the device,the cell cluster can be encapsulated by the semipermeable membrane. Theencapsulation can be performed by any technique available to one skilledin the art. The semipermeable membrane can also be made of any suitablematerial as one skilled in the art would appreciate and verify. Forexample, the semipermeable membrane can be made of polysaccharide orpolycation. In some cases, the semipermeable membrane can be made ofpoly(lactide) (PLA), poly(glycolic acid) (PGA),poly(lactide-co-glycolide) (PLGA), and other polyhydroxyacids,poly(caprolactone), polycarbonates, polyamides, polyanhydrides,polyphosphazene, polyamino acids, polyortho esters, polyacetals,polycyanoacrylates, biodegradable polyurethanes, albumin, collagen,fibrin, polyamino acids, prolamines, alginate, agarose, agarose withgelatin, dextran, polyacrylates, ethylene-vinyl acetate polymers andother acyl-substituted cellulose acetates and derivatives thereof,polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride,poly(vinyl imidazole), chlorosulphonated polyolefins, polyethyleneoxide, or any combinations thereof. In some cases, the semipermeablemembrane comprises alginate. In some cases, the cell cluster isencapsulated in a microcapsule that comprises an alginate coresurrounded by the semipermeable membrane. In some cases, the alginatecore is modified, for example, to produce a scaffold comprising analginate core having covalently conjugated oligopeptides with an RGDsequence (arginine, glycine, aspartic acid). In some cases, the alginatecore is modified, for example, to produce a covalently reinforcedmicrocapsule having a chemoenzymatically engineered alginate of enhancedstability. In some cases, the alginate core is modified, for example, toproduce membrane-mimetic films assembled by in-situ polymerization ofacrylate functionalized phospholipids. In some cases, microcapsules arecomposed of enzymatically modified alginates using epimerases, In somecases, microcapsules comprise covalent links between adjacent layers ofthe microcapsule membrane. In some embodiment, the microcapsulecomprises a subsieve-size capsule comprising alginate coupled withphenol moieties. In some cases, the microcapsule comprises a scaffoldcomprising alginate-agarose. In some cases, the SC-β cell is modifiedwith PEG before being encapsulated within alginate. In some cases, theisolated populations of cells, e.g., SC-β cells are encapsulated inphotoreactive liposomes and alginate. It should be appreciated that thealginate employed in the microcapsules can be replaced with othersuitable biomaterials, including, without limitation, polyethyleneglycol (PEG), chitosan, polyester hollow fibers, collagen, hyaluronicacid, dextran with ROD, BHD and polyethylene glycol-diacrylate (PEGDA),poly(MPC-co-n-butyl methacrylate-co-4-vinylphenyl boronic acid) (PMBV)and poly(vinyl alcohol) (PVA), agarose, agarose with gelatin, andmultilayer cases of these. In some cases, the device provided hereincomprise extracorporeal segment, e.g., part of the device can be outsidea subject's body when the device is implanted in the subject. Theextracorporeal segment can comprise any functional component of thedevice, with or without the cells or cell cluster provided herein.

Methods of Treatment

Further provided herein are methods for treating or preventing a diseasein a subject. A composition comprising the cell clusters or cellsprovided herein or generated according to the methods provided hereincan be administered into a subject to restore a degree of pancreaticfunction in the subject. For example, the cell clusters resemblingendogenous pancreatic islets, or the cells resembling endogenouspancreatic α, β and/or δ cells (e.g., non-native pancreatic α, β and/orδ cells) or the precursors thereof can be transplanted to a subject totreat diabetes. Most typically, a composition to be administered into asubject comprises cells that are fully differentiated, or cells that arenearly fully differentiated. However, as further differentiation ofcells can be achieved in vivo, the present disclosure is not limited inthis respect. For example, in some embodiments, a composition to beencapsulated in a device and/or administered into a subject comprisescells that are not fully differentiated (e.g., a composition comprisingPDX1-positive, NKX6.1-negative pancreatic progenitor cells, andPDX1-positive, NKX6.1-positive pancreatic progenitor cells).

The methods can comprise transplanting the cell cluster or the celldisclosed in the application to a subject, e.g., a subject in needthereof. The term “transplanting” can refer to the placement of cells orcell clusters, any portion of the cells or cell clusters thereof, or anycompositions comprising cells, cell clusters or any portion thereof,into a subject, by a method or route which results in at least partiallocalization of the introduced cells or cell clusters at a desired site.The cells or cell clusters can be implanted directly to the pancreas, oralternatively be administered by any appropriate route which results indelivery to a desired location in the subject where at least a portionof the implanted cells or cell remain viable. The period of viability ofthe cells or cell clusters after administration to a subject can be asshort as a few hours, e.g. twenty-four hours, to a few days, to as longas several years. In some instances, the cells or cell clusters, or anyportion of the cells or cell clusters thereof, can also betransadministered at a non-pancreatic location, such as in the liver orsubcutaneously, for example, in a capsule (e.g., microcapsule) tomaintain the implanted cells or cell clusters at the implant locationand avoid migration.

As used herein, the term “treating” and “treatment” can refer toadministering to a subject an effective amount of a composition (e.g.,cell clusters or a portion thereof) so that the subject as a reductionin at least one symptom of the disease or an improvement in the disease,for example, beneficial or desired clinical results. For purposes ofthis disclosure, beneficial or desired clinical results include, but arenot limited to, alleviation of one or more symptoms, diminishment ofextent of disease, stabilized (e.g., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (e.g., partial or total), whetherdetectable or undetectable. Treating can refer to prolonging survival ascompared to expected survival if not receiving treatment. Thus, one ofskill in the art realizes that a treatment may improve the diseasecondition, but may not be a complete cure for the disease. As usedherein, the term “treatment” includes prophylaxis.

Exemplary modes of administration include, but are not limited to,injection, infusion, instillation, inhalation, or ingestion. “Injection”includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebrospinal, and intrasternal injection and infusion. Inpreferred embodiments, the compositions are administered by intravenousinfusion or injection.

By “treatment,” “prevention” or “amelioration” of a disease or disorderis meant delaying or preventing the onset of such a disease or disorder,reversing, alleviating, ameliorating, inhibiting, slowing down orstopping the progression, aggravation or deterioration the progressionor severity of a condition associated with such a disease or disorder.In one embodiment, the symptoms of a disease or disorder are alleviatedby at least 5%, at least 10%, at least 20%, at least 30%, at least 40%,or at least 50%.

Treatment of Diabetes is determined by standard medical methods. A goalof Diabetes treatment is to bring sugar levels down to as close tonormal as is safely possible. Commonly set goals are 80-120 milligramsper deciliter (mg/dl) before meals and 100-140 mg/dl at bedtime. Aparticular physician may set different targets for the patent, dependingon other factors, such as how often the patient has low blood sugarreactions. Useful medical tests include tests on the patient's blood andurine to determine blood sugar level, tests for glycosylated hemoglobinlevel (HbA1c; a measure of average blood glucose levels over the past2-3 months, normal range being 4-6%), tests for cholesterol and fatlevels, and tests for urine protein level. Such tests are standard testsknown to those of skill in the art (see, for example, American DiabetesAssociation, 1998). A successful treatment program can also bedetermined by having fewer patients in the program with complicationsrelating to Diabetes, such as diseases of the eye, kidney disease, ornerve disease.

Delaying the onset of diabetes in a subject refers to delay of onset ofat least one symptom of diabetes, e.g., hyperglycemia, hypoinsulinemia,diabetic retinopathy, diabetic nephropathy, blindness, memory loss,renal failure, cardiovascular disease (including coronary arterydisease, peripheral artery disease, cerebrovascular disease,atherosclerosis, and hypertension), neuropathy, autonomic dysfunction,hyperglycemic hyperosmolar coma, or combinations thereof, for at least 1week, at least 2 weeks, at least 1 month, at least 2 months, at least 6months, at least 1 year, at least 2 years, at least 5 years, at least 10years, at least 20 years, at least 30 years, at least 40 years or more,and can include the entire lifespan of the subject.

In some aspects, the disclosure relates to a method comprisingimplanting in a subject a device comprising a cell or cell clusterprovided herein (e.g., insulin producing cells), wherein the devicereleases insulin in an amount sufficient for a reduction of bloodglucose levels in the subject. In some embodiments, the insulinproducing cells are glucose responsive insulin producing cells.

In some embodiments, the reduction of blood glucose levels in thesubject, as induced by the transplantation of the cell or cell cluster,or the device provided herein, results in an amount of glucose which islower than the diabetes threshold. In some embodiments, the subject is amammalian subject. In some embodiments, the mammalian subject is human.In some embodiments, the amount of glucose is reduced to lower than thediabetes threshold in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after theimplanting.

As described in detail above, the pharmaceutical compositions of thepresent disclosure can be specially formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), lozenges, dragees, capsules, pills, tablets(e.g., those targeted for buccal, sublingual, and systemic absorption),boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation; (3) topical application,for example, as a cream, ointment, or a controlled-release patch orspray applied to the skin; (4) intravaginally or intrarectally, forexample, as a pessary, cream or foam; (5) sublingually; (6) ocularly;(7) transdermally; (8) transmucosally; or (9) nasally. Additionally,compounds can be implanted into a patient or injected using a drugdelivery system. See, for example, Urquhart, et al., Ann. Rev.Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. “Controlled Releaseof Pesticides and Pharmaceuticals” (Plenum Press, N.Y., 1981); U.S. Pat.No. 3,773,919; and U.S. Pat. No. 35 3,270,960.

A subject that can be treated by the methods herein can be a human or anon-human animal. In some cases, a subject can be a mammal. Examples ofa subject include but are not limited to primates, e.g., a monkey, achimpanzee, a bamboo, or a human. In some cases, a subject is a human. Asubject can be non-primate animals, including, but not limited to, adog, a cat, a horse, a cow, a pig, a sheep, a goat, a rabbit, and thelike. In some cases, a subject receiving the treatment is a subject inneed thereof, e.g., a human in need thereof.

In certain embodiments, the subject is a mammal, e.g., a primate, e.g.,a human. The terms, “patient” and “subject” are used interchangeablyherein. Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of Type 1diabetes, Type 2 Diabetes Mellitus, or pre-diabetic conditions. Inaddition, the methods described herein can be used to treat domesticatedanimals and/or pets. A subject can be male or female. A subject can beone who has been previously diagnosed with or identified as sufferingfrom or having Diabetes (e.g., Type 1 or Type 2), one or morecomplications related to Diabetes, or a pre-diabetic condition, andoptionally, but need not have already undergone treatment for theDiabetes, the one or more complications related to Diabetes, or thepre-diabetic condition. A subject can also be one who is not sufferingfrom Diabetes or a pre-diabetic condition. A subject can also be one whohas been diagnosed with or identified as suffering from Diabetes, one ormore complications related to Diabetes, or a pre-diabetic condition, butwho show improvements in known Diabetes risk factors as a result ofreceiving one or more treatments for Diabetes, one or more complicationsrelated to Diabetes, or the pre-diabetic condition. Alternatively, asubject can also be one who has not been previously diagnosed as havingDiabetes, one or more complications related to Diabetes, or apre-diabetic condition. For example, a subject can be one who exhibitsone or more risk factors for Diabetes, complications related toDiabetes, or a pre-diabetic condition, or a subject who does not exhibitDiabetes risk factors, or a subject who is asymptomatic for Diabetes,one or more Diabetes-related complications, or a pre-diabetic condition.A subject can also be one who is suffering from or at risk of developingDiabetes or a pre-diabetic condition. A subject can also be one who hasbeen diagnosed with or identified as having one or more complicationsrelated to Diabetes or a pre-diabetic condition as defined herein, oralternatively, a subject can be one who has not been previouslydiagnosed with or identified as having one or more complications relatedto Diabetes or a pre-diabetic condition.

The methods can comprise transplanting the cell cluster to a subjectusing any means in the art. For example the methods can comprisetransplanting the cell cluster via the intraperitoneal space, renalsubcapsule, renal capsule, omentum, subcutaneous space, or viapancreatic bed infusion. For example, transplanting can be subcapsulartransplanting, intramuscular transplanting, or intraportaltransplanting, e.g., intraportal infusion. Immunoprotectiveencapsulation can be implemented to provide immunoprotection to the cellclusters. In some cases, the methods of treatment provided herein cancomprise administer immune response modulator for modulating or reducingtransplant rejection response or other immune response against theimplant (e.g., the cells or the device). Examples of immune responsemodulator that can be used in the methods can include purine synthesisinhibitors like Azathioprine and Mycophenolic acid, pyrimidine synthesisinhibitors like Leflunomide and Teriflunomide, antifolate likeMethotrexate, Tacrolimus, Ciclosporin, Pimecrolimus, Abetimus,Gusperimus, Lenalidomide, Pomalidomide, Thalidomide, PDE4 inhibitor,Apremilast, Anakinra, Sirolimus, Everolimus, Ridaforolimus,Temsirolimus, Umirolimus, Zotarolimus, Anti-thymocyte globulinantibodies, Anti-lymphocyte globulin antibodies, CTLA-4, fragmentthereof, and fusion proteins thereof like Abatacept and Belatacept, TNFinhibitor like Etanercept and Pegsunercept, Aflibercept, Alefacept,Rilonacept, antibodies against complement component 5 like Eculizumab,anti-TNF antibodies like Adalimumab, Afelimomab, Certolizumab pegol,Golimumab, Infliximab, and Nerelimomab, antibodies against Interleukin 5like Mepolizumab, anti-Ig E antibodies like Omalizumab, anti-Interferonantibodies like Faralimomab, anti-IL-6 antibodies like Elsilimomab,antibodies against IL-12 and IL-23 like Lebrikizumab and Ustekinumab,anti-IL-17A antibodies like Secukinumab, anti-CD3 antibodies likeMuromonab-CD3, Otelixizumab, Teplizumab, and Visilizumab, anti-CD4antibodies like Clenoliximab, Keliximab, and Zanolimumab, anti-CD11aantibodies like Efalizumab, anti-CD18 antibodies like Erlizumab,anti-CD20 antibodies like Obinutuzumab, Rituximab, Ocrelizumab andPascolizumab, anti-CD23 antibodies like Gomiliximab and Lumiliximab,anti-CD40 antibodies like Teneliximab and Toralizumab, antibodiesagainst CD62L/L-selectin like Aselizumab, anti-CD80 antibodies likeGaliximab, anti-CD147/Basigin antibodies like Gavilimomab, anti-CD154antibodies like Ruplizumab, anti-BLyS antibodies like Belimumab andBlisibimod, anti-CTLA-4 antibodies like Ipilimumab and Tremelimumab,anti-CAT antibodies like Bertilimumab, Lerdelimumab, and Metelimumab,anti-Integrin antibodies like Natalizumab, antibodies againstInterleukin-6 receptor like Tocilizumab, anti-LFA-1 antibodies likeOdulimomab, antibodies against IL-2 receptor/CD25 like Basiliximab,Daclizumab, and Inolimomab, antibodies against T-lymphocyte (Zolimomabaritox) like Atorolimumab, Cedelizumab, Fontolizumab, Maslimomab,Morolimumab, Pexelizumab, Reslizumab, Rovelizumab, Siplizumab,Talizumab, Telimomab aritox, Vapaliximab, and Vepalimomab.

“Antifoaming agents” reduce foaming during processing which can resultin coagulation of aqueous dispersions, bubbles in the finished film, orgenerally impair processing. Exemplary anti-foaming agents includesilicon emulsions or sorbitan sesquoleate.

“Antioxidants” include, for example, butylated hydroxytoluene (BHT),sodium ascorbate, ascorbic acid, sodium metabisulfite and tocopherol. Incertain embodiments, antioxidants enhance chemical stability whererequired.

Formulations described herein may benefit from antioxidants, metalchelating agents, thiol containing compounds and other generalstabilizing agents. Examples of such stabilizing agents, include, butare not limited to: (a) about 0.5% to about 2% w/v glycerol, (b) about0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/vmonothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% toabout 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate80, (g) 0.001% to about 0.05% w/v. polysorbate 20, (h) arginine, (i)heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosanpolysulfate and other heparinoids, (m) divalent cations such asmagnesium and zinc; or (n) combinations thereof.

“Binders” impart cohesive qualities and include, e.g., alginic acid andsalts thereof; cellulose derivatives such as carboxymethylcellulose,methylcellulose (e.g., Methocel®), hydroxypropylmethylcellulose,hydroxyethylcellulose, hydroxypropylcellulose (e.g., Klucel®),ethylcellulose (e.g., Ethocel®), and microcrystalline cellulose (e.g.,Avicel®); microcrystalline dextrose; amylose; magnesium aluminumsilicate; polysaccharide acids; bentonites; gelatin;polyvinylpyrrolidone/vinyl acetate copolymer; crospovidone; povidone;starch; pregelatinized starch; tragacanth, dextrin, a sugar, such assucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol,xylitol (e.g., Xylitab®), and lactose; a natural or synthetic gum suchas acacia, tragacanth, ghatti gum, mucilage of isapol husks,polyvinylpyrrolidone (e.g., Polyvidone® CL, Kollidon® CL, Polyplasdone®XL-10), larch arabogalactan, Veegum®, polyethylene glycol, waxes, sodiumalginate, and the like.

A “carrier” or “carrier materials” include any commonly used excipientsin pharmaceutics and should be selected on the basis of compatibilitywith compounds disclosed herein, such as, compounds of ibrutinib and Ananticancer agent, and the release profile properties of the desireddosage form. Exemplary carrier materials include, e.g., binders,suspending agents, disintegration agents, filling agents, surfactants,solubilizers, stabilizers, lubricants, wetting agents, diluents, and thelike. “Pharmaceutically compatible carrier materials” may include, butare not limited to, acacia, gelatin, colloidal silicon dioxide, calciumglycerophosphate, calcium lactate, maltodextrin, glycerine, magnesiumsilicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters,sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine,sodium chloride, tricalcium phosphate, dipotassium phosphate, celluloseand cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, and the like. See,e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed(Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins1999).

“Dispersing agents,” and/or “viscosity modulating agents” includematerials that control the diffusion and homogeneity of a drug throughliquid media or a granulation method or blend method. In someembodiments, these agents also facilitate the effectiveness of a coatingor eroding matrix. Exemplary diffusion facilitators/dispersing agentsinclude, e.g., hydrophilic polymers, electrolytes, Tween ® 60 or 80,PEG, polyvinylpyrrolidone (PVP; commercially known as Plasdone®), andthe carbohydrate-based dispersing agents such as, for example,hydroxypropyl celluloses (e.g., HPC, HPC-SL, and HPC-L), hydroxypropylmethylcelluloses (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M),carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate,hydroxypropylmethylcellulose acetate stearate (HPMCAS), noncrystallinecellulose, magnesium aluminum silicate, triethanolamine, polyvinylalcohol (PVA), vinyl pyrrolidone/vinyl acetate copolymer (S630),4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol), poloxamers (e.g., PluronicsF68®, F88®, and F108®, which are block copolymers of ethylene oxide andpropylene oxide); and poloxamines (e.g., Tetronic 908®, also known asPoloxamine 908®, which is a tetrafunctional block copolymer derived fromsequential addition of propylene oxide and ethylene oxide toethylenediamine (BASF Corporation, Parsippany, N.J.)),polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetatecopolymer (S-630), polyethylene glycol, e.g., the polyethylene glycolcan have a molecular weight of about 300 to about 6000, or about 3350 toabout 4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, polysorbate-80, sodium alginate, gums, such as, e.g.,gum tragacanth and gum acacia, guar gum, xanthans, including xanthangum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose,methylcellulose, sodium carboxymethylcellulose, poly sorbate-80, sodiumalginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitanmonolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates,chitosans and combinations thereof. Plasticizers such as cellulose ortriethyl cellulose can also be used as dispersing agents. Dispersingagents particularly useful in liposomal dispersions and self-emulsifyingdispersions are dimyristoyl phosphatidyl choline, natural phosphatidylcholine from eggs, natural phosphatidyl glycerol from eggs, cholesteroland isopropyl myristate.

Combinations of one or more erosion facilitator with one or morediffusion facilitator can also be used in the present compositions.

The term “diluent” refers to chemical compounds that are used to dilutethe compound of interest prior to delivery. Diluents can also be used tostabilize compounds because they can provide a more stable environment.Salts dissolved in buffered solutions (which also can provide pH controlor maintenance) are utilized as diluents in the art, including, but notlimited to a phosphate buffered saline solution. In certain embodiments,diluents increase bulk of the composition to facilitate compression orcreate sufficient bulk for homogenous blend for capsule filling. Suchcompounds include e.g., lactose, starch, mannitol, sorbitol, dextrose,microcrystalline cellulose such as Avicel®; dibasic calcium phosphate,dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate;anhydrous lactose, spray-dried lactose; pregelatinized starch,compressible sugar, such as Di-Pac® (Amstar); mannitol,hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetatestearate, sucrose-based diluents, confectioner's sugar; monobasiccalcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactatetrihydrate, dextrates; hydrolyzed cereal solids, amylose; powderedcellulose, calcium carbonate; glycine, kaolin; mannitol, sodiumchloride; inositol, bentonite, and the like.

“Filling agents” include compounds such as lactose, calcium carbonate,calcium phosphate, dibasic calcium phosphate, calcium sulfate,microcrystalline cellulose, cellulose powder, dextrose, dextrates,dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol,mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

“Lubricants” and “glidants” are compounds that prevent, reduce orinhibit adhesion or friction of materials. Exemplary lubricants include,e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, ahydrocarbon such as mineral oil, or hydrogenated vegetable oil such ashydrogenated soybean oil (Sterotex®), higher fatty acids and theiralkali-metal and alkaline earth metal salts, such as aluminum, calcium,magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes,Stearowet®, boric acid, sodium benzoate, sodium acetate, sodiumchloride, leucine, a polyethylene glycol (e.g., PEG-4000) or amethoxypolyethylene glycol such as Carbowax™, sodium oleate, sodiumbenzoate, glyceryl behenate, polyethylene glycol, magnesium or sodiumlauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sil®, a starchsuch as corn starch, silicone oil, a surfactant, and the like.

“Plasticizers” are compounds used to soften the microencapsulationmaterial or film coatings to make them less brittle. Suitableplasticizers include, e.g., polyethylene glycols such as PEG 300, PEG400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propyleneglycol, oleic acid, triethyl cellulose and triacetin. In someembodiments, plasticizers can also function as dispersing agents orwetting agents.

“Solubilizers” include compounds such as triacetin, triethylcitrate,ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate,vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone,N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropylalcohol, cholesterol, bile salts, polyethylene glycol 200-600,glycofurol, transcutol, propylene glycol, and dimethyl isosorbide andthe like.

“Stabilizers” include compounds such as any antioxidation agents,buffers, acids, preservatives and the like.

“Suspending agents” include compounds such as polyvinylpyrrolidone,e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17,polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e.g.,the polyethylene glycol can have a molecular weight of about 300 toabout 6000, or about 3350 to about 4000, or about 7000 to about 5400,sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate,polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as,e.g., gum tragacanth and gum acacia, guar gum, xanthans, includingxanthan gum, sugars, cellulosics, such as, e.g., sodiumcarboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80,sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylatedsorbitan monolaurate, povidone and the like.

“Surfactants” include compounds such as sodium lauryl sulfate, sodiumdocusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitanmonooleate, polyoxyethylene sorbitan monooleate, polysorbates,polaxomers, bile salts, glyceryl monostearate, copolymers of ethyleneoxide and propylene oxide, e.g., Pluronic® (BASF), and the like. Someother surfactants include polyoxyethylene fatty acid glycerides andvegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40. In some embodiments, surfactants may be included toenhance physical stability or for other purposes.

“Viscosity enhancing agents” include, e.g., methyl cellulose, xanthangum, carboxymethyl cellulose, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetatestearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinylalcohol, alginates, acacia, chitosans and combinations thereof.

“Wetting agents” include compounds such as oleic acid, glycerylmonostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamineoleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate,sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium saltsand the like.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1 Application of PKC Activator For Differentiation of PancreaticEndocrine Cells

This example demonstrates the effect of PKC activator on generation ofdifferent pancreatic endocrine cells, e.g., pancreatic β, α, δ, or ECcell.

Exemplary differentiation protocols, e.g., Version A and Version B,according to the present disclosure were tested for differentiatinghuman stem cells into mature β cells capable of releasing insulin inresponse to glucose challenge in vitro.

Both Version A and Version B protocols are 6-stage stepwise protocolsthat share similar reagents and treatment timing. With Version Aprotocols, stem cells were treated with reagents in the followingconsecutive orders during the first five stages: Stage 1 (51), Activin-Afor 3 days and also CHIR99021 for the first 24 hr; Stage 2 (S2), KGF for3 days; Stage 3 (S3), KGF, PDBU, Sant-1, retinoic acid (RA), Activin A,and Thiazovivin for 2 days, and also DMH-1 for the first day; Stage 4(S4), KGF, Sant-1, Thiazovivin, Activin A, and RA for 6 days; Stage 5(S5), XXI, Alk5i, GC-1, LDN-193189, Thiazovivin, Staurosporine, andDZNEP for 7 days, and also RA, Sant-1, and Betacellulin for the first 2days. In Version B protocols, one difference from Version A protocols isthat the cells were supplemented with PDBU from day 5 of Stage 4 (S4d5)to day 2 of Stage 5 (S5d2). In one experiment, 500 nM of PDBU was usedto treat the cells during S4d5 to S5d2.

As illustrated in the schematics of single-cell RNA sequencing resultsin FIG. 1, upon completion of S5 differentiation, the cells (S5c cells)generated via a Version B protocol and S5c cells generated via a VersionA protocol had comparable percentage of cells expressing CHGA (geneencoding chromogranin A; an exemplary marker of pancreatic endocrinecells), and comparable percentage of cells expressing ISL1 (an exemplarymarker of pancreatic islet cells). In contrast, S5c cells generated viaVersion B protocol had much reduced percentage of cells expressing DDC(gene encoding dopa decarboxylase; an exemplary marker ofenterochromaffin (EC) cells).

In one experiment, four different exemplary Stage 6 treatment paradigmswere also tested at S6 in combination with the first five stages ofVersion A or Version B protocols. Briefly, i) with S6-a paradigm, S5ccells were cultured in DMEM/F12 medium containing 1% HSA for 7-14 days;ii) with S6-b paradigm, S5c cells were cultured in MCDB131 mediumcontaining 0.05% HSA and the following supplements (per 1L MCDB131):0.44 g Glucose, 1.23 g NaHCO3, 0.044 g Vitamin C, 10 ml Glutamax, and 5ml ITS-x; iii) with S6-c paradigm, S5c cells were cultured in MCDB131medium containing 0.05% HSA and vitamin C for 7-14 days, during whichthe cells were treated with 10 μM Alk5i, 1 μM GC-1, 100 nM LDN-193189,2.5 μM Thiazovivin, 3 nM SSP, and 100 nM DZNEP for the first four days;iv) with S6-d paradigm, S5c cells were cultured in MCDB131 medium (noglutamine base media) containing 0.05% HSA, ITS-X, vitamin C, and 4 mMGln, as well as 10 μM Alk5i, 1 μM GC-1, 100 nM LDN-193189, 2.5 μMThiazovivin, 3 nM SSP, and 100 nM DZNEP, for four days, followed byculturing in MCDB131 medium (no glutamine base media) containing 0.05%HSA, ITS-X, vitamin C, and 4 mM Gln with no additional factors foradditional 3-10 days.

FIG. 2A demonstrates the increase in percentage of cells that expressC-peptide and do not express VMAT1 (exemplary characteristic of Sc-βcells) generated by Version B protocols as compared to Version Aprotocols, as measured by flow cytometry (FIG. 2B). As shown in thefigure, with Version A protocols, the percentage of C-peptide-positive,VMAT1-negative cells on S6d9 or S6d14 were all around 35%, except forVA/S6-a and VA/S6-d on S6d9 (both around 40%), whereas with Version Bprotocols, the percentage of C-peptide-positive, VMAT1-negative cellswas from 45% to 55% on S6d9, and around 45% on S6d14.

FIGS. 3-5 demonstrate the changes in percentages of α cells (measured bycells that express glucagon (GCG) but do not express somatostatin (SST)via flow cytometry), δ cells (measured by cells that express SST but donot express GCG via flow cytometry), and EC cells (measured by cellsthat express VMAT1 but do not express C-peptide via flow cytometry) withVersion B protocols as compared to Version A protocols. As shown in FIG.3, with Version A protocols, the percentage of α cells on S6d9 or S6d14was all below or around 5%, whereas with Version B protocols, thepercentage of α cells was from 12.5% to 17% on S6d9, and around 15%(vB/S6-c and vB/S6-d) or around 22% (vB/S6-a and vB/S6-b) on S6d14. Asshown in FIG. 4, with vA protocols, the percentage of δ cells on S6d9 orS6d14 was below or around 2% (vA/S6-a and vA/S6-b) or from 2% to 3%(vA/S6-c and vA/S6-d), whereas with Version B protocols, the percentageof δ cells was around 5% on S6d9 and S6d14 (vB/S6-a), around 7% on S6d9and around 5% on S6d14 (vB/S6-b), from 6% to 7% on S6d9 and S6d14(vB/S6-c), or around 8% on S6d9 and S6d14 (vB/S6-d). As shown in FIG. 5,with Version A protocols, the percentage of EC cells on S6d9 or S6d14was from 40% to 50% (vA/S6-a), from 35% to 45% (vA/S6-b), or from 25% to35% (vA/S6-c and vA/S6-d), whereas with vB protocols, the percentage ofEC cells was all less than 20% on S6d9 and S6d14.

In one experiment, SOX9 expression was compared between Version A andVersion B protocols, and it was found that at the end of S5, there wereincreased cells expressing SOX9 with Version B protocols as compared toVersion A protocols (FIG. 6A). In another experiment, a reaggregationstep was introduced between the end of S5 and the beginning of S6.Briefly, S5c cell clusters were collected and dissociated into cellsuspension with an enzyme and then cultured in S6 culture media toreaggregate into new cell clusters. FIG. 6B summarizes the percentage ofcells expressing SOX9 during S6 after the reaggregation step withdifferent differentiation protocols. In another experiment, the S6 cellrecovery percentage was examined, which measured the ratio of the celldensity at a certain time point of S6 (e.g., S6d4, S6d9, or S6d14)relative to the initial seed density at the beginning of S6 (afterdissociation of S5c cells, e.g., 2 million/ml). As shown in FIG. 7,Version B protocols had similar S6 cell recovery percentage as comparedto Version A protocols.

In one experiment, in vitro glucose-stimulated insulin secretionresponse of S6d13 cells generated by Version A and Version B protocolswas examined. As shown in FIG. 8, the cells generated with Version Bprotocols showed relatively low responsiveness to glucose challenges,and there was no clear increase in insulin secretion in response to 20mM glucose challenge as compared to 2.8 mM glucose challenge. Incontrast, cells generated with vA/S6-a protocol demonstrated sharpincrease in insulin secretion in response to 20 mM as compared to 2.8 mMglucose challenge. On the other hand, insulin content in cells generatedwith Version B protocols was comparable with cells generated withVersion B protocols (FIG. 9).

In one experiment, effect of application of PDBU and gamma secretaseinhibitor, XXI, was tested. In this experiment, three differentiationconditions were examined and compared: Version A protocol; Version B;Version B+ XXI (both PDBU and XXI was applied from S4d5 to S5d2, and XXIcontinued to be applied throughout S5). FIG. 10B summarizes thepercentages of ISL1-positive cells and ISL1-negative cells at S5cgenerated via different protocols, as measured by flow cytometry(exemplified in FIG. 10A). As shown in the figure, combined PKCactivation and gamma secretase inhibition starting during S4 led torobust induction of ISL1-positive cells from about 65% to higher than90%, whereas PKC activation alone led to about 80% ISL1-positive cells.

In another experiment, the effect of two different PKC activators onenterochromaffin cells and alpha cells was examined. In this experiment,three differentiation conditions were examined and compared: Version Aprotocol; Version A with PDBU applied at 0.5 μM on days S4d5, S5d1, andS5d2 (VA+PDBU); and Version A with((2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam)at 0.1 μM on days S4d5, S5d1, and S5d2 (VA+TPPB). FIG. 11B summarizesthe percentages of NKX6.1-positive/ISL-negative cells andISL1-positive/NKX6.1-negative cells at S5c generated via the differentprotocols, as measured by flow cytometry (exemplified in FIG. 11A). Asshown in the figure, VA/TPPB was similarly effective as VA/PDBU atreducing sc-EC cells and increasing alpha cells. However, VA/TPPBsurprisingly resulted in a more than 2-fold increase in yield of totalcells as compared to VA/PDBU (FIG. 11C). Addition of XXI to VA/TPPB atdays S4d5, S5d1, and S5d2 caused a reduction in in cell yield (FIG.11C).

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of the presentdisclosure can be employed in practicing the present disclosure. It isintended that the following claims define the scope of the presentdisclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

1.-30. (canceled)
 31. A method, comprising: (a) contacting a pluralityof PDX1-positive, NKX6.1-negative pancreatic progenitor cells with oneor more of a ROCK inhibitor, a growth factor from TGFβ superfamily, agrowth factor from FGF family, a RA signaling pathway activator, and aSHH pathway inhibitor, thereby generating a first population of cells;(b) contacting the first population of cells with a PKC activator and aγ-secretase inhibitor and one or more of a ROCK inhibitor, a growthfactor from the TGFβ superfamily, a growth factor from the FGF family, aRA signaling pathway activator, and a SHH pathway inhibitor, therebygenerating a second population of cells; and (c) contacting the secondpopulation of cells with a PKC activator, a γ-secretase inhibitor andone or more of a TGF-β signaling pathway inhibitor, a growth factor fromEGF family, a RA signaling pathway activator, a SHH pathway inhibitor, aTH signaling pathway activator, a protein kinase inhibitor, a ROCKinhibitor, a BMP signaling pathway inhibitor, and an epigeneticmodifying compound, thereby generating a third population of cells.32.-89. (canceled)
 90. An in vitro composition, comprisingPDX1-positive, NKX6.1-negative pancreatic progenitor cells;PDX1-positive, NKX6.1-positive pancreatic progenitor cells; a PKCactivator; and a γ-secretase inhibitor. 91.-136. (canceled)
 137. An invitro composition comprising PDX1-positive cells, a γ-secretaseinhibitor, and one or both of a growth factor from the TGFβ superfamilyand a growth factor from the FGF family. 138.-166. (canceled)
 167. Apopulation of in vitro differentiated cells comprising NKX6.1-positive,ISL1-positive cells and NKX6.1-negative, ISL1-positive cells; andwherein less than 12% of the cells in the population areNKX6.1-negative, ISL1-negative cells.
 168. The population of claim 167,wherein less than 10%, less than 8%, less than 6%, or less than 4% ofthe cells in the population are NKX6.1-negative, ISL1-negative cells.169. The population of claim 167, wherein at least 60%, at least 65%, atleast 70%, at least 73%, at least 75%, or at least 80% of the cells inthe population are ISL1-positive cells.
 170. The population of claim167, wherein 2-12%, 4-12%, 6-12%, 8-12%, 2-8%, 4-8%, 3-6% or 3-5% of thecells in the population are NKX6.1-negative, ISL1-negative cells. 171.The population of claim 167, wherein 50-90%, 50-85%, 50-80%, 50-75%,50-70%, 50-60%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 65-90%, 65-85%,65-80%, 65-75%, 65-70%, 70-90%, 70-85%, 70-80%, 70-75%, 75-90%, 75-85%,75-80%, 80-90%, 80-85%, or 85-90% of the cells in the population areISL1-positive cells.
 172. (canceled)
 173. The population of claim 167,wherein at least 4%, at least 5%, at least 6%, at least 8%, at least10%, about 4-11%, or about 5-10% of the cells in the population areNKX6.1-negative, ISL1-negative cells.
 174. The population of claim 167,wherein at least 40% of the cells in the population are NKX6.1-negative,ISL1-positive cells.
 175. The population of claim 167, wherein at least45%, at least 50%, about 40-50%, about 45-55%, or about 50-55% of thecells in the population are NKX6.1-negative, ISL1-positive cells. 176.The population of claim 167, wherein at least 74%, at least 75%, atleast 80%, at least 85%, at least 90%, about 85-95%, or about 90-95% ofthe cells in the population are ISL1-positive cells.
 177. (canceled)178. The population of claim 167, wherein the population of cells isderived from stem cells in vitro.
 179. The population of claim 167,further comprising a medium.
 180. The population of claim 179, whereinthe medium comprises a sugar.
 181. (canceled)
 182. The population ofclaim 180, wherein the medium comprises the sugar at a concentration ofbetween about 0.05% and about 1.5%.
 183. The population of claim 179,wherein the medium is a CMRL medium; or wherein the medium isHypoThermosol® FRS Preservation Media.
 184. The population of claim 167,wherein the population of cells is in a cell cluster.
 185. Thepopulation of claim 167, wherein the population of cells are in one ormore cell cluster.
 186. The population of claim 185, wherein the cellcluster is between about 125 and about 225 microns in diameter, betweenabout 130 and about 160 microns in diameter, between about 170 and about225 microns in diameter, between about 140 and about 200 microns indiameter, between about 140 and about 170 microns in diameter, betweenabout 160 and about 220 microns in diameter, between about 170 and about215 microns in diameter, or between about 170 and about 200 microns indiameter.
 187. The population of claim 167, wherein the population has agenetic disruption in the beta-2-microglobulin gene.
 188. The populationof claim 167, wherein the population comprises NKX6.1-positive,ISL1-positive cells that express lower levels of MAFA thanNKX6.1-positive, ISL1-positive cells from the pancreas of a healthycontrol adult subject.
 189. The population of claim 167, wherein thepopulation comprises NKX6.1-positive, ISL1-positive cells that expresshigher levels of MAFB than NKX6.1-positive, ISL1-positive cells from thepancreas of a healthy control adult subject.
 190. The population ofclaim 167, wherein the population comprises NKX6.1-positive,ISL1-positive cells that express higher levels of SIX2, HOPX, IAPPand/or UCN3 than NKX6.1-positive, ISL1-positive cells from the pancreasof a healthy control adult subject. 191.-193. (canceled)
 194. Animplantable encapsulation device comprising the population of claim 167.195. -196. (canceled)
 197. A method of treating a subject, the methodcomprising administering to the subject a composition comprising thepopulation of claim 167.